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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Environmental Microbial & Food Safety Laboratory » Research » Research Project #430681

Research Project: Antimicrobial Resistance and Ecology of Zoonotic Foodborne Pathogens in Dairy Cattle

Location: Environmental Microbial & Food Safety Laboratory

2019 Annual Report

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.

Progress Report
Substantial progress has been made towards the research goals for each of the three objectives during the third year of the project. For 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 (~1800) have been determined and the data are currently being analyzed. Additionally, a controlled calf experiment was started in April, 2019 to evaluate the impact of feeding waste milk that contains residual antibiotics on resistance in enteric bacteria of calves from birth until post-weaning. Age at weaning will also be evaluated for impact on antimicrobial resistance profiles in post-weaned calves. Calves have been sourced from a single commercial herd and the dams are sampled at the time of pick-up (within 24 h of birth). Calves are individually housed without the ability to contact each other. Fecal samples are collected daily for 1 week followed by weekly thereafter. All samples are cultured for E. coli and Salmonella and isolates are tested for resistance to a panel of 11 antibiotics. Additionally, samples from each calf at each time point are being preserved for metagenomic analyses. Because it is too expensive to sequence the metagenome of every sample, the samples will be selected for this purpose based on the results of the antimicrobial resistance analysis of the bacterial isolates. The animal component of this experiment will take approximately nine months but metagenome sample collection will begin after the first group of animals have cycled through the pens (August 2019). These experiments will be complemented with another project evaluating the presence of antibiotic resistance in in utero calves of culled dams at slaughter along with the birth canals of the dams to determine if any transmission of resistant bacteria occurs before birth. To further study potential factors that are involved in colonization of young calf intestines with antimicrobial-resistant E. coli, a panel of 26 E. coli strains from dairy calves representing multiple phylogenetic groups and diverse antimicrobial resistance patterns has been assembled from our culture collection. The panel will be used in multiple experiments so that comparisons can be made across experiments. 1.) The panel is being used to compare the growth rates diverse E. coli strains in media that is supplemented with bovine colostrum. The hypothesis is that growth rates of isolates from different phylogenetic groups and different resistance phenotypes will not be the same. Colostrum was sourced from a commercial dairy herd and frozen in quantities that could be used for each assay and a pasteurization technique was developed that would retain the consistency of the colostrum. Because of the opacity and consistency of the colostrum, significant effort was required to develop approaches to minimize variability in the assay; the assays are nearing completion. 2.) A cell culture assay has been developed to determine the capacity of E. coli strains to bind or attach to an immortalized bovine epithelial cell-line. The hypothesis is that binding capacity of isolates from different phylogenetic groups and different resistance phenotypes will not be the same. The technique has been tested with control and test strains and the analysis of the strains in the select E. coli panel is ongoing. In a project focused on evaluating potential influences of lysogenic phages as inserts into the genomes of Gram-negative bacteria on the virulence, metabolism, and antimicrobial sensitivity in their bacterial hosts, preliminary analyses of dairy-associated bacterial genomes identified phage proteins in nearly all the genomes. To effectively study the potential effects of phage-bacterial interactions on antimicrobial resistance, we selected a lysogen (Salmonella enterica serovar Typhimurium) that harbored bacteriophage P22 and developed a non-lysogen by continual sub-culture and screening. We then compared several physicochemical properties, e.g., growth, hydrophobicity, auto-aggregation, biofilm formation, and cellular motility, between the lysogen and the non-lysogen. We detected significant differences in growth rate and cellular motility, but no differences were observed in hydrophobicity, auto-aggregation, or biofilm formation capabilities. We have sequenced these isolates to determine whether bacteriophage P22 inserts into S. Typhimurium genome at random or at specific insertion site; analyses of the data are currently in progress. Phenotypic microarrays using Biolog PM plates (PM1 to PM20) are being used to identify differences in antimicrobial susceptibility and metabolic potential between the lysogen and the non-lysogen. The first trial has been run and data analyses is in progress. Antimicrobials that will show different effects on lysogen versus non-lysogen will be screened and bacterial growth kinetics will be studied in the presence of selected antimicrobials. For Objective 2: To identify the non-resistance-conferring genomic features in calf-associated multi-drug resistant (MDR) E. coli that facilitate the colonization of the gut of newborn calves 254 MDR and pan-susceptible E. coli genomes from lactating cows, dry cows, post-weaned calves, and pre-weaned calves were sequenced. To focus on the non-resistance conferring elements the genomes were interrogated for the presence of 340 virulence-associated genes, plasmid types, and protein-coding genes. Our analysis shows that a significant number of virulence genes were detected in E. coli from all four groups and plasmid profiles were highly diverse. An analysis of protein-coding genes showed that there were over 15,000 genes in the pan-genome of E. coli collected from these animals. Currently these data are being analyzed to determine if any virulence-associated genes, plasmids, and protein-coding genes are more enriched in isolates from any of the four animal groups, resulting in a selective colonization advantage, with an emphasis on persistence in the calf gut. Based on these data, pre-weaned calves will be inoculated with MDR and pan-susceptible E. coli to evaluate the efficacy of non-resistance conferring genomic features in persistence in the calf gut. For Objective 3: To analyze the ability of Salmonella serotypes that behave as commensal inhabitants of the dairy cow gut and serotypes that are transient in the cow (or cause systemic infections) to interact with cultured bovine cells, an immortalized bovine epithelial cell-line was used for an association-invasion assay. Comparisons of the attachment and invasion capacities of four strains of S. Cerro, and two strains from each of S. Anatum, S. Dublin, S. Give, S. Kentucky, S. Mbandaka, S. Meleagridis, S. Montevideo, S. Muenster, S. Newport, S. Oranienberg, S. Senftenberg, and S. Typhimurium serotypes have been made. Results indicated significant differences in the ability of the Salmonella strains to interact with bovine cells based on serotype. To identify potential factors that contributed to different propensity of cellular invasion by different Salmonella serotypes, protocols for characterizing transcriptomes of Salmonella serotypes during invasion are being developed. Because this is a new area of research, two members of our lab group attended training to learn metagenomic analysis approaches and techniques. Initially, the transcriptome of one S. Dublin strain has been sequenced in duplicate to identify the sequencing depth that will be required to effectively compare differentially expressed transcripts across the serotypes. Based on the analysis, the transcriptome of a subset of strains will be characterized during invasion into bovine cells. Interactions of dairy animal-associated Salmonella enterica with bovine epithelial cells. Salmonella is a leading cause of bacterial diarrhea worldwide and dairy cattle are known reservoirs of this zoonotic pathogen. While some serotypes cause disease in cows and calves, others are often shed from animals showing no symptoms of the infection and, in these cases, contamination of the food products or the surrounding environment may go unnoticed. To explore the mechanism of how individual serotypes colonize dairy cattle, ARS scientists at Beltsville, Maryland, infected bovine epithelial cells (in the laboratory) with Salmonella strains from different serotypes and observed a significant difference in the ability of the Salmonella strains to interact (attach and/or invade) with bovine cells based on serotype. For example, serotype Dublin, which can cause salmonellosis in cows, was more likely to invade bovine cells than other serotypes that are frequently shed by healthy cows, and Salmonella Newport strains, which are commonly associated with sick cows, showed an invasion pattern similar to that of other serotypes that are not known to cause severe disease in cattle. These results suggest that colonization of Salmonella serotypes in dairy cows are more complex than previously thought, and further research is needed to understand these dynamics and develop ways to reduce the carriage of Salmonella in dairy cows.

1. Multidrug-resistant Escherichia coli from veal calves. Dairy animals can carry antibiotic-resistant bacteria that impact both human and animal health; however, data on antibiotic-resistant bacteria shed in the feces of veal calves is very limited. ARS scientists at Beltsville, Maryland, analyzed the genomes of 155 E. coli samples collected from the feces of veal calves and identified more than 1000 antibiotic resistance genes, including some resistant to aminoglycoside antibiotics, an important group of antibiotics in human medicine. Fifteen E. coli antibiotic-resistant samples have the potential to cause urinary infections in humans. Scientists will use this knowledge to help the veal industry to further understand the antimicrobial resistance E. coli.

2. Salmonella in dairy and beef cattle in the United States. Salmonella (S. Dublin) is a significant human pathogen that is mainly carried by beef and dairy cattle globally and the incidence of human infections in the United States has risen in recent years. ARS scientists at Beltsville, Maryland, collaborated with ARS scientists in Clay Center, Nebraska, surveyed the distribution of salmonella in dairy and beef in the United States and abroad. When the genetics of 107 samples collected from 24 countries were compared to those collected in the United States, they were different. In the United States, salmonella was found in five major areas. Results of this study will help scientists, dairy farmers and ranchers across the United States, and will the tracking of outbreaks.

Review Publications
Springer, H., Denagamage, T., Fenton, G., Haley, B.J., Van Kessel, J.S., Hovingh, E. 2019. Antimicrobial resistance in fecal Escherichia coli and Salmonella enterica in dairy calves: a systematic review. Foodborne Pathogens and Disease. 16:23-24.
Salaheen, S., Kim, S., Karns, J.S., Hovingh, E., Haley, B.J., Van Kessel, J.S. 2019. Metagenomic analysis of fecal microbiome in Escherichia coli O157:H7-shedding and non-shedding animals from a single dairy farm. Food Control. 102:76-80.
Cao, H., Pradhan, A.K., Karns, J.S., Wolfgang, D.R., Hovingh, E., Van Kessel, J.S., Vinyard, B.T., Kim, S. 2019. Age-associated distribution of antimicrobial-resistant Salmonella enterica and Escherichia coli isolated from dairy herds in Pennsylvania, 2013-2015. Foodborne Pathogens and Disease. 16:60-67.
Callahan, M., Van Kessel, J.S., Micallef, S.A. 2018. Salmonella enterica recovery from river waters of the Maryland Eastern Shore reveals high serotype diversity and some multidrug resistance. Environmental Research. 168:7-13.
Salaheen, S., Kim, S., Cao, H., Wolfgang, D., Karns, J., Haley, B.J., Hovingh, E., Van Kessel, J.S. 2019. Antimicrobial resistance among Escherichia coli isolated from veal calf operations in Pennsylvania. Foodborne Pathogens and Disease. 16:74-80.
Salaheen, S., Cao, H., Sonnier, J.L., Kim, S., Del Collo, L.P., Hovingh, E., Karns, J.S., Haley, B.J., Van Kessel, J.S. 2019. Diversity of extended-spectrum cephalosporin-resistant Escherichia coli in feces from calves and cows on Pennsylvania dairy farms. Foodborne Pathogens and Disease.
Salaheen, S., Kim, S., Karn, J.S., Haley, B.J., Van Kessel, J.S. 2018. Fecal metagenome sequences from lactating dairy cows shedding Escherichia coli O157:H7. Microbiology Resource Announcements. 7:e01279-18.
Haley, B.J., Smith, T.P., Harhay, G.P., Loneragan, G.H., Webb, H.E., Bugarel, M., Kim, S., Van Kessel, J.S., Harhay, D.M. 2019. Complete genome sequence of a Salmonella enterica subsp. enterica serovar Fresno isolate recovered from beef cattle lymph nodes. Microbiology Resource Announcements. 8(2):e01338-18.