Impact of intramuscular ceftiofur treatment on the gut microbiome  and resistome of dairy cattle
Third-generation cephalosporins such as ceftiofur, are classified as critically important antibiotics because pathogens with resistance to these drugs contribute to high mortality rates and can be readily transmitted between individuals. Furthermore, these antibiotics are frequently administered in animals, emphasizing the crucial role they play in both human and veterinary medicine. Because metritis is a common condition requiring antibiotic treatment in dairy cattle, we sought to investigate the impact of intramuscular ceftiofur treatment on the abundance of resistant bacteria and resistance genes in the fecal microbiome. Twelve cows with metritis (cases) were enrolled and treated with ceftiofur for comparison to 12 matched untreated cows (controls). Fecal samples were collected from both groups prior to ceftiofur treatment of the cases and weekly for up to four weeks. The quantity of Gram-negative bacteria with resistance to ceftiofur, ampicillin, and tetracycline was determined using MacConkey lactose agar, while metagenomic next-generation sequencing was used to characterize the microbiome and resistome. Importantly, the treated cases had persistently higher levels of Gram-negative bacteria with resistance to ceftiofur but not to ampicillin or tetracycline. Persistently higher levels of genes encoding extended-spectrum beta-lactamase (ESBL) production were also observed in the treated cows from weeks 1-4. blaCMY-4, blaACI-1, cfxA5, and cfxA6 were the most abundant genes detected. Although no substantial changes in microbiota diversity were observed, some taxa differed in abundance post-treatment. Moreover, the abundance and diversity of plasmids and virulence genes were lower one week after treatment. Bacteroidetes and Escherichia coli were identified as the main bacterial hosts of ESBL genes, while an analysis of beta-lactamase-carrying contigs revealed the co-localization of ESBLs with plasmid and transposase genes, indicating their potential involvement in the horizontal transfer of these resistance genes. Together, these findings demonstrate the persistent effects of ceftiofur treatment on the abundance of resistant bacterial populations and ESBL genes in dairy cattle over a short time frame. Implementing strategies to limit the use of ceftiofur and other critically important antibiotics can be vital for preserving the efficacy of these antibiotics and safeguarding public and animal health.