Bovines are the most important source of protein, and their production is expected to intensify with the increased human population. Despite the benefits of cattle, their intensification can result in increased negative footprints such as the transmission of foodborne pathogens and antibiotic-resistant (AR) bacteria. Cattle are the primary carriers of Shiga toxin-producing Escherichia coli (STEC) which causes over 250,000 human infections in the U.S. every year. Furthermore, about half of the mass of antibiotics sold in the U.S. are used in bovines. Third-generation cephalosporins are commonly used in cattle (i.e., ceftiofur); however, these antibiotics are considered of last resort. Globally, Enterobacteriaceae resistant to third-generation cephalosporins are the main cause of death by AR infections. In this dissertation three aims were addressed: 1) identifying microbiome diversity and composition that may favor the colonization of STEC in the hindgut of cattle; 2) determining the impacts of intramammary (IMM) ceftiofur in dairy cattle in the abundance of AR bacteria in feces; and 3) characterizing changes in the functional microbiome and metabolome associated with the IMM ceftiofur application.In Chapter 2, we analyzed 660 fecal samples from beef and dairy cattle from 5 farms with varying prevalences of STEC. The microbiome composition analyzed with 16S rRNA gene sequencing revealed that the microbiota of animals from farms with a high-STEC prevalence (HSP) had greater richness compared to those of farms with a low-STEC prevalence (LSP). Higher microbiome diversity was also identified in STEC-shedders from LSP farms but not in animals from HSP farms. Finally, we evidenced that bacterial taxa associated with STEC shedding in dairy farms were also correlated with differences in the diet and risk factors of STEC carriage such as days in milk, number of lactations, and warm temperatures. In Chapter 3, we evaluated the effects of intramammary (IMM) ceftiofur application on the abundance of resistant bacteria in cattle feces. Twenty dairy cows were treated with IMM ceftiofur and a non-antibiotic internal teat sealant and another group of 20 cows (controls) received only the non-antibiotic sealant. Feces were collected the day before the treatment and in weeks 1, 2, 3, 5, 7, and 9 after the treatment (n = 278). Through culture-based methods, no differences were observed in the number of β-lactam resistant bacteria (i.e., ampicillin and ceftiofur) between treatment groups. However, metagenomic sequencing revealed a greater abundance of genes encoding extended-spectrum β-lactamases (ESBL) that confer resistance to third-generation cephalosporins. Furthermore, an increased number of correlations between β-lactam resistance genes, mobile genetic elements, and bacterial genera were observed a week after IMM ceftiofur treatment. Finally, in Chapter 4 we analyzed the effects of IMM ceftiofur in the functional microbiome and metabolome of feces collected in the prior chapter. The IMM antibiotic treatment had minor effects on the functional microbiome, while no differences were observed in the metabolome between treatment groups. Multi-omics analyses identified correlations between natural antimicrobial compounds, pesticides, bacteriophages from enterobacteria, and ESBL genes. This suggests the role of natural bacterial stressors in the abundance of MGEs and ARGs. This dissertation aims to provide information for reducing the prevalence of STEC and antibiotic resistance in cattle farms, which is critical to prevent infections in humans and ensure the effectiveness of last-resort treatments.
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