Objective 1: Examine the relationship between gut bacteria and the bovine host to determine factors that contribute to observed age-related differences in colonization by AMR bacteria. 1A: Determine the capacity of resistant E. coli strains to bind or attach to intestinal epithelial cells. 1B: Evaluate and compare the growth rates of resistant E. coli strains in media that is supplemented with bovine colostrum or milk replacer. 1C: Examine the developing microbial community structure in the young calf intestine and the ability of resistant E. coli strains to outcompete other strains/species in these communities. Objective 2: Examine and determine if resistance determinants in bacteria are linked to specific genomic characteristics that influence bacterial colonization capacity in the young dairy calf. 2A: Identify non-resistance conferring genomic features in calf-associated MDR E. coli that facilitate the colonization of the gut of newborn calves. 2B: Examine the ability of generic, susceptible E. coli strains to outcompete MDR E. coli strains in the gut of newborn calves. Objective 3: Compare and contrast interactions between bovine host cells and Salmonella enterica to identify factors that contribute to differences between Salmonella serotypes that behave as commensal inhabitants of the dairy cow gut and serotypes that are transient in the cow or cause systemic infections.
Although the products of American dairy farms are overwhelmingly safe, food producing animals are known reservoirs for bacteria that are detrimental to human health and outbreaks have been attributed to consumption of contaminated raw milk, raw milk products, or meat. Additionally, the impact of animal production on the burden of antibiotic resistant bacteria affecting humans has become a major issue although the contribution of dairy farming to this burden is currently unknown. This project is composed of three major objectives relating to bacteria of public health importance that are associated with dairy animals. Resistant bacteria are more prevalent in dairy calves than in cows and multi-drug resistant bacteria are often found in pre-weaned calves. We will take a three-pronged approach to study resistance in dairy calves. We will investigate interactions between resistant E. coli and intestinal epithelial cells, relationships between resistant E. coli and the developing gut community, and associations between resistance determinants and genomic characteristics that influence bacterial colonization capacity in the calf. This project also builds on previous work characterizing the ecology of bacterial pathogens in dairy animals by determining factors associated with the establishment and maintenance of infections in cows. We will analyze the ability of Salmonella strains to bind to bovine epithelial cells and relate observed differences in binding and gene expression to factors responsible for the persistence of commensal-type Salmonella serotypes in dairy cows. We will compare the interactions of these serotypes with host intestinal cells with the interactions of serotypes that are transient in the cow or cause systemic infections in dairy cows. The project will improve our understanding of antibiotic resistance in dairy calves and commensal Salmonella infections in dairy cows so that new approaches for mitigation can be developed.
Substantial progress has been made towards the research goals for each of the three objectives during the fourth year of the project. Objective 1: Two animal experiments in collaboration with scientists at Pennsylvania State University are ongoing. The goal of the first experiment is to compare the antibiotic resistance profiles of E. coli shed in the feces of dairy calves that were raised under different management approaches (diet, antibiotic treatment, weaning age). Fecal samples have been collected from calves on 13 commercial herds and E. coli were isolated from each sample. The antibiotic resistance profiles of the E. coli isolates have been determined and the data have been analyzed. Based on the results, a subset of fecal samples were selected and the metagenomes from these samples were sequenced and are currently being analyzed. Analysis of the microbial community and the resistomes are on-going. Additionally, a controlled calf experiment was conducted to evaluate the impact of feeding waste milk that contains residual antibiotics on resistance in enteric bacteria of calves from birth until post-weaning. Calves were individually housed so that there was no contact with each other. Fecal samples were collected daily for 1 week followed by weekly thereafter for up to 2 weeks after weaning. The study took nearly one year to complete. All samples were cultured for E. coli and Salmonella and isolates are being tested for resistance to a panel of 11 antibiotics. The resistance testing has been delayed due to the COVID pandemic; however, it is anticipated that this portion of the project will be completed by December 2020. Objective 2: We’ve compared growth rates and characterized the attachment and invasion properties of susceptible and AR E. coli and no differences were observed in growth rates or the capacity of AR and susceptible E. coli to associate with bovine epithelial cells. However, we did observe significant differences between E. coli strains in their ability to associate with bovine epithelial cells. This led us to look for additional genetic factors that may be responsible for a lack of an apparent association between resistance determinants and interaction with bovine epithelial cells. The genomes of 288 E. coli isolates from different age groups across 80 dairy herds were sequenced and annotated. Statistical analysis of the pangenomes of the multi drug resistant (MDR) and susceptible isolates identified over 500 accessory genes that were more abundant in MDR than susceptible isolates. Annotation of these genes revealed that some are potential targets for the mitigation of MDR carriage in dairy animals, including iron scavenging genes and myo-inositol transport and metabolism genes, among many others. Work is ongoing to confirm this finding and to determine the potential significance of these genomic differences with respect to carriage of antimicrobial resistant bacteria in calves. Objective 3: As a follow-up to a recently completed accomplishment describing the differences in the abilities of common dairy cow-associated Salmonella serovars to attach to and invade bovine epithelial, we are currently conducting a transcriptomic analyses of Salmonella Dublin and Cerro strains in association with bovine epithelial cells. The goal of this is to understand the genomic features that allow some serovars to cause severe or fatal infections in cows while others persist as commensal members of the gut community. To-date, we have optimized a method to separate host cells from bacteria before RNA extraction. Further, we also had to address the inefficiency of the lysis buffer in commercial RNA extraction kit by increasing the bead-beating time and treating the samples with RNase inhibitor before RNA extraction. We are currently optimizing the removal of ribosomal RNA from total RNA in order to efficiently sequence mRNA which will allow us to evaluate gene expression and ultimately the genomic factors involved in serovar-specific infection and colonization.
1. Antibiotic resistance in Escherichia coli isolated from bovine. ARS scientists at Beltsville, Maryland, collaborated with scientists at Eduardo Mondlane University in Maputo, Mozambique, and the University of Sassari, Italy, to recover resistant E. coli from bovine feces in and around Maputo, Mozambique. Resistance to tetracycline and ampicillin was observed with very low levels of multi-drug resistance. The results of this study help to further our understanding of antimicrobial-resistant bacteria associated with food-producing animals in different production environments and enhanced the technical capacity of food scientists and veterinarians in Mozambique.
2. Metagenomic analysis of the fecal microbial communities and resistomes of veal calves. Farm animals are known to harbor antimicrobial-resistant bacteria, including human pathogens, sometimes irrespective of recent antimicrobial use and we previously observed that antimicrobial-resistant E. coli were widespread in the feces from veal calves at all ages. To evaluate the prevalence and types of antibiotic resistance genes, the metagenomes (all genes from all organisms) of fecal samples from 12 veal calves that just arrived on their farm and 12 calves approximately 3 months later, were sequenced to identify the microbial communities and resistance genes found therein. Results confirmed previous culture-based analysis and demonstrated high prevalences of resistance-conferring bacterial genes in veal calf feces, including those that many confer resistance to medically important antimicrobials. The data indicate that veal calf feces can harbor a diverse suite of antibiotic resistance genes, some of which are clinically significant to human health. More information is needed to understand the factors that contribute to antimicrobial-resistant bacteria in veal calves and to determine management factors that could be adopted to reduce the prevalence of resistance at the animal and farm level.
3. Antimicrobial-resistant fecal E. coli populations in dairy calves. The use of antimicrobials in adult cows and in the young calves on dairy farms carry the highest prevalence of antimicrobial-resistant (AMR) E. coli. ARS scientists in Beltsville, Maryland, collaborated with researchers at Pennsylvania State University to evaluate how AMR E. coli populations change as calves age. They found that about 90 percent of the samples from the preweaned calves contained AMR E. coli. The early postnatal period is a critical time for colonization of the calf gut by resistant bacteria, and weaning plays a key role in reducing resistance in the calf gut. The study informs scientists and industry of potential targets for mitigating the prevalence of AMR bacteria harbored in young dairy calves.
Haley, B.J., Kim, S., Salaheen, S., Hovingh, E., Van Kessel, J.S. 2020. Differences in the microbial community and resistome structures of feces from pre-weaned calves and lactating dairy cows. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2019.2768.
Salaheen, S., Sonnier, J.L., Kim, S., Haley, B.J., Van Kessel, J.S. 2020. Interaction of Salmonella enterica with bovine epithelial cells demonstrates serovar-specific attachment and invasion patterns. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2019.2765.
Haley, B.J., Kim, S., Haendiges, J., Keller, E., Torpey, D., Kim, A., Crocker, K., Myers, R.A., Van Kessel, J.S. 2019. Salmonella enterica serovar Kentucky recovered from human clinical cases in Maryland, USA (2011 to 2015). Journal of Clinical Microbiology. 66:382-392. https://doi.org/10.1111/zph.12571.