Ensiling of forages was recognized as a microbial-driven process as early as the late 1800s, when it was associated with the production of "sweet" or "sour" silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage.

Escherichia coli O157:H7 is a foodborne pathogen that causes illness in humans worldwide. Cattle are the primary reservoir of this bacterium, with the concentration and frequency of E. coli O157:H7 shedding varying greatly among individuals. The term "super-shedder" has been applied to cattle that shed concentrations of E. coli O157:H7 ≥ 10⁴ colony-forming units/g feces. Super-shedders have been reported to have a substantial impact on the prevalence and transmission of E. coli O157:H7 in the environment. The specific factors responsible for super-shedding are unknown, but are presumably mediated by characteristics of the bacterium, animal host, and environment. Super-shedding is sporadic and inconsistent, suggesting that biofilms of E. coli O157:H7 colonizing the intestinal epithelium in cattle are intermittently released into feces. Phenotypic and genotypic differences have been noted in E. coli O157:H7 recovered from super-shedders as compared to low-shedding cattle, including differences in phage type (PT21/28), carbon utilization, degree of clonal relatedness, tir polymorphisms, and differences in the presence of stx2a and stx2c, as well as antiterminator Q gene alleles. There is also some evidence to support that the native fecal microbiome is distinct between super-shedders and low-shedders and that low-shedders have higher levels of lytic phage within feces. Consequently, conditions within the host may determine whether E. coli O157:H7 can proliferate sufficiently for the host to obtain super-shedding status. Targeting super-shedders for mitigation of E. coli O157:H7 has been proposed as a means of reducing the incidence and spread of this pathogen to the environment. If super-shedders could be easily identified, strategies such as bacteriophage therapy, probiotics, vaccination, or dietary inclusion of plant secondary compounds could be specifically targeted at this subpopulation. Evidence that super-shedder isolates share a commonality with isolates linked to human illness makes it imperative that the etiology of this phenomenon be characterized.

Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed “super-shedders”. A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a “normal” microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.

Escherichia coli strains isolated from fecal samples were screened to examine changes in phenotypic and genotypic characteristics including antimicrobial susceptibility, clonal type, and carriage of resistance determinants. The goal of this 197-day study was to investigate the influence of administration of chlortetracycline alone (T) or in combination with sulfamethazine (TS) on the development of resistance, dissemination of defined strain types, and prevalence of resistance determinants in feedlot cattle. Inherent tetracycline resistance was detected in cattle with no prior antimicrobial exposure. Antimicrobial administration was not found to be essential for the maintenance of inherently ampicillin-resistant and tetracycline-resistant (Tet r ) E. coli in control animals; however, higher Tet r E. coli shedding was observed in animals subjected to the two treatments. At day 0, high tetracycline (26.7%), lower sulfamethoxazole-tetracycline (19.2%), and several other resistances were detected, which by the finishing phase (day 197) were restricted to ampicillin-tetracycline (47.5%), tetracycline (31.7%), and ampicillin-tetracycline-sulfamethoxazole (20.8%) from both treated and untreated cattle. Among the determinants, bla TEM1 , tet(A), and sul2 were prevalent at days 0 and 197. Further, E. coli from day 0 showed diverse antibiogram profiles and strain types, which by the finishing phase were limited to up to three, irrespective of the treatment. Some genetically identical strains expressed different phenotypes and harbored diverse determinants, indicating that mobile genetic elements contribute to resistance dissemination. This was supported by an increased linked inheritance of ampicillin and tetracycline resistance genes and prevalence of specific strains at day 197. Animals in the cohort shed increasingly similar genotypes by the finishing phase due to animal-to-animal strain transmission. Thus, characterizing inherent resistance and propagation of cohort-specific strains is crucial for determining antimicrobial resistance in cattle.In recent years, the development of antimicrobial resistance (AR) in intensive livestock production has been increasingly reported (17). Consequently, the role of antimicrobial administration and the extent to which it affects the development of resistance in animals are receiving much attention. Antimicrobial usage for livestock can be for therapeutic, prophylactic, metaphylactic, or growth promotion purposes (26). Reportedly, 90% of the antimicrobials used in animal agriculture are for growth promotion and prophylaxis (26) and this widespread use is suggested to be an important contributor to the emergence, selection, and dissemination of antimicrobial-resistant bacteria, as indicated by several recent studies (9, 13, 28, 38). Antimicrobial-resistant bacteria, including Escherichia coli, are frequently isolated from the commensal gut flora of food animals (1, 2), and although the resistance they carry may not be a problem per se, the transfer of resistance elements to zoonotic...