Perturbations in Intestinal Antibiotic-resistant Bacterial Populations in Dairy Cattle Following Intramammary Treatment With a Third-generation Cephalosporin

Background. The impact of antimicrobials used to treat and prevent diseases in livestock requires careful monitoring, particularly for those drugs considered to be critically important in human medicine. Such is the case for third-generation cephalosporins commonly used in cattle farms. Use of these drugs may select for antibiotic-resistant (AR) bacteria including extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, the most economically impactful antimicrobial-resistant threat. Hence, we sought to evaluate the impact of intramammary (IMM) treatment with ceftiofur on the cattle-gut microbiome and abundance of resistant bacteria. Methods. Forty healthy dairy cows were enrolled at the end of lactation: 20 were treated with IMM ceftiofur (SpectramastDC) and a non-antibiotic internal teat sealant (bismuth subnitrate) and 20 received only bismuth subnitrate as a control. Fecal samples were collected from each cow before (day -1) and after treatment (weeks 1, 2, 3, 5, 7, and 9) for quantification of bacteria with resistance to beta-lactams as well as metagenomic next-generation sequencing (mNGS). Gram-negative and Gram-positive bacteria were quantified on MacConkey-lactose and Columbia CNA agar, respectively, and initial mNGS and bioinformatic analyses were done using CosmosID (Germantown, MD 20874) pipelines. Results. One week after dry-off, there were no differences in quantity of total or AR bacteria between cows treated with IMM ceftiofur and controls. Although there was no difference in prevalence of bacteria resistant to beta-lactams between treatment groups, 90% and 24% of samples analyzed (n = 278) had Gram-negative bacteria that were resistant to ampicillin and ceftiofur, respectively. On average, the number of Gram-negative ceftiofur-resistant bacteria increased up to 2 weeks in cows treated with antibiotic, but not significant due to high variation among animals, with 25% shedding more ceftiofur-resistant bacteria. Cows treated with IMM ceftiofur had lower microbiome richness during weeks 5 and 9, characterized by decreased abundance of Proteobacteria, Tenericutes, Verrucomicrobia, and Firmicutes and higher abundance of Bacteroidetes and Actinobacteria and greater abundance of ESBL (bla-CFXA, bla-ACI-1, and bla-CMY) and blc genes throughout the 9-week period (LDA > 3.3; P < 0.03), namely Enterobacteriaceae and commensal bacteria. Network analysis also showed persistent increase in correlations between beta-lactam resistant genes and phages, MGEs, and bacterial genera during weeks 1 and 9 post IMM ceftiofur treatment. Conclusion. This study highlights long-term effects of 2 g IMM ceftiofur given at dry off on the gut microbiome and resistome composition in dairy cattle. Despite a single IMM administration of a third-generation cephalosporin, treated cattle showed increased abundance of genes encoding ESBLs in feces, persisting for 9 weeks. Commensal bacteria were associated predominantly with carriage of ESBLs and, in lower proportion, with Enterobacteriaceae. Shedding of ESBL-Enterobacteriaceae was variable among animals in that 75% did not show increased number of ceftiofur-resistant bacteria after IMM antibiotic application. Additional research should identify associations between fecal shedding and the potential for dissemination and persistence of antibiotic resistance determinants in dairy farms.