1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Substantial progress has been made towards the research goals for each of the three objectives: A protocol for determining growth rates of E. coli in milk replacer has been developed. The primary challenges have been ensuring reproducibility and increasing the number of strains that can be run in a single experiment. These objectives are exacerbated by the opaqueness of the media and the need to enumerate via plating vs. optical density. An analysis of the resistance of E. coli isolates from calves raised in different herds under different conditions has been initiated in collaboration with scientists at Penn State University. More than 1500 isolates have been isolated from calf fecal samples that were collected from 13 herds. The resistance phenotypes are being characterized for each sample. The E. coli resistance profiles of will be compared between calves of different ages and between calves that were being reared under differing management approaches. An experimental protocol was established to compare the ability of different strains of Salmonella enterica to attach and invade bovine cells. Initial comparisons of the attachment and invasion capacities of 32 S. Kentucky strains that were isolated from different sources have been made. Additionally, strains representing 12 different Salmonella serotypes have been tested and the data are being processed. Development of approach and protocols for characterizing the transcriptome of the bacterial cells during the attachment and invasion process has begun. Lysogenic phages insert into the genomes of Gram-negative bacteria and potentially influence the virulence, metabolism, and sensitivity to antibiotics of that bacterium. Preliminary analyses of dairy-associated bacterial genomes were conducted and phage proteins were identified in nearly all of the genomes. We’ve begun a project to pursue the potential for collateral prophage-bacterial interactions that may have a significant impact on antimicrobial resistance due to antibiotic use in food animals. Salmonella Heidelberg has caused severe and fatal infections in humans and is frequently isolated from poultry in the United States. A recent outbreak, in which young children were predominantly affected, of multi-drug resistant S. Heidelberg was linked to dairy calves in Wisconsin. We were asked by individuals in APHIS and at the Wisconsin Department of Agriculture to assist with identifying ways to test for S. Heidelberg. They were particularly interested in our milk filter sampling approach to test for the presence of this particularly virulent zoonotic bacteria on dairy farms. We are planning to evaluate the presence of S. Heidelberg in a subset of Wisconsin dairy herds by testing milk filters using traditional culture-based methods and direct detection methods. As part of this work novel real-time PCR detection methods are being developed to specifically detect S. Heidelberg and discriminate against all other sequenced serovars. In collaboration with other ARS scientists and university partners, a metagenomic analysis of the bovine rumen microbial community is being conducted. To evaluate the composition of this community and investigate the biases of different sequencing methods, a single rumen sample was collected and sequenced using two different sequencing chemistries. The two sequencing runs were separately assembled and the presence of antimicrobial resistance genes is being assessed in both assemblies. Analysis is on-going. Multiple evolutionary lineages, known as sequence types (STs), of Salmonella Kentucky exist and these STs are presumed to have significantly different levels of virulence in humans. For example, ST152, which is predominant in North American food animals, appears to be relatively benign to humans, while ST198, which is predominant outside of the Americas, appears to cause severe infections and is frequently MDR. Both STs are found in animals and uncooked food products in the United States. To better understand the differences between the STs, we are comparing the genomes of all six ST lineages. PCR primers are also being developed to identify the ST of S. Kentucky isolates. This will aid in monitoring the potential emergence of ST198 in food-producing animals in North America. We have continued analyzing the genomes of Salmonella Dublin from bovine, human, and other sources. Salmonella Dublin is a bovine-adapted serovar and, although S. Dublin is a low prevalence serovar in human infections, hospitalization and mortality rates are higher for Dublin infections vs. more common serovars. Additionally, S. Dublin isolates are frequently multidrug resistant. Genomes sequenced in-house were analyzed alongside publicly available genomes. Current work is focused on detecting specific sequences that are associated with invasive infections in cows. As part of this project real-time PCR methods previously developed will be improved upon.
1. Resistome and community analysis of pre-weaned dairy calves and lactating dairy cows. Although the role of dairy cattle in the transmission of these bacteria to humans is unclear, they are reservoirs of antibiotic resistant bacteria; in addition; dairy calves harbor more resistant bacteria than adult cows. ARS scientists at Beltsville, Maryland, sequenced the genomes of the bacteria (metagenome) found in the feces of pre-weaned dairy calves and lactating dairy cows from 12 farms. Results indicated that feces of pre-weaned dairy calves have a significantly different bacterial community population than lactating cows. Similarly, the resistomes (the totality of antibiotic resistance genes in the bacteria) were significantly different between the adults and calves. The relative abundance of antibiotic resistance genes was highest in feces from the younger animals. Results of this analysis support the observation that dairy animals are colonized with antimicrobial resistant bacteria at a very young age and indicates that more information is needed to determine the factors that affect this early colonization in order to determine mitigation approaches that dairy operations could use to potentially decrease the abundance of antibiotic resistance in these animals.
2. Interactions of Salmonella Kentucky, a commensal member of the bovine intestinal microbial community with bovine epithelial cells. S. Kentucky, a distinct variation of Salmonella, is frequently isolated from the feces of dairy cows and multiple lineages (distantly related organisms) with differing genome compositions and virulence genes, have been isolated from food-producing animals in North America. Only the lineage known as ST152 is predominant in dairy cows and variations have been observed within this lineage in their ability to persist within an individual dairy herd. To identify the potential factors involved in this disproportionate presence in dairy cows in North America and persistence within a herd, ARS scientists at Beltsville, Maryland, artificially infected bovine mammary epithelial cells in culture, as a proxy for bovine intestinal epithelial cells, with a set of S. Kentucky isolates from cows as well as multiple varieties from a long-term S. Kentucky outbreak in a dairy herd. Results indicated a decrease in the invasiveness of S. Kentucky isolated over time from within the individual herd-level outbreak and a difference in the abilities of lineages to attach and invade into the bovine cells, suggesting multiple factors may be involved in the selection of ST152 in dairy cows in the United States. Because this contrasts with food-producing animals in Europe, South Asia, and Africa where ST198 is the predominant type, this research suggests external factors, such as management practices or geography, may play a role in the bovine carriage of this zoonotic pathogen.
3. Age-dependent differences in the antimicrobial resistance among the gut microbial communities of veal calves. Previous studies have demonstrated that antimicrobial resistance was more prevalent in younger farm animals compared to their older counterparts. This study used cutting-edge techniques to investigate age-associated differences in the microbial communities found in feces and antimicrobial resistance gene composition (resistome) in veal calves. The results showed a clear difference in the microbial community structure and the resistomes of different aged veal calves. Observations from this study may help to better manage detrimental antimicrobial resistance occurrence in dairy animals.
4. Differences in antibiotic-susceptible and multi-drug resistant (MDR) Escherichia (E.) coli isolated from pre-weaned calves and lactating dairy cows. The presence, abundance and types of antibiotic resistance in E. coli that are isolated from the feces of food-producing animals is often used as a proxy for the overall presence, abundance, and type of antibiotic resistance in the intestinal microbiomes of these animals, including dairy animals. Previous research demonstrated that pre-weaned calves have more antibiotic resistant E. coli than older animals. ARS scientists at Beltsville, Maryland, have compared the genomes of 160 E. coli isolates collected from two animal groups. Results indicate that most E. coli isolates are phylogenetic group B1, are less likely to be MDR, and that older animals are more likely to carry these B1 isolates than younger animals. E. coli isolates from pre-weaned calves were more likely to include more virulence genes than those from lactating cows. This indicates that pre-weaned calves are more likely to carry MDR E. coli and might also carry E. coli that are more likely to cause disease than older lactating cows. The results of this study establish a better understanding of the differences between the E. coli populations of young calves and adult cows.
Kim, S., Haley, B.J., Roberson, D., Allard, M., Hammack, T., Brown, E.W., Van Kessel, J.S. 2017. The Genome sequences of four non-human/non-clinical Salmonella enterica serovar Kentucky ST198 isolates recovered between 1972 and 1973. Genome Announcements. https://doi.org/10.1128/genomeA.01699-16.
Salaheen, S., Kim, S., Haley, B.J., Van Kessel, J.S., Biswas, D. 2017. Alternative growth promoters alter broiler gut microbiome and enhance body weight gain. Front. Microbiol.8:2088. BMC Microbiome. https://doi.org/10.3389/fmicb.2017.02088.
Kim, S., Karns, J.S., Van Kessel, J.S., Haley, B.J. 2017. Genome sequences of thirty Escherichia coli O157:H7 isolates recovered from a single dairy farm and its associated off-site heifer raising facility. Genome Announcements. https://doi.org/10.1128/genomeA.00814-17.
Sonnier, J.L., Karns, J.S., Lombard, J.E., Kopral, C.A., Haley, B.J., Kim, S., Van Kessel, J.S. 2018. Prevalence of Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli in bulk tank milk and milk and milk filters from U.S. dairy operations in the National Animal Health Monitoring System Dairy 2014 study. Journal of Dairy Science. 101:1943-1956. https://doi.org/10.3168/jds.2017-13546.
Kim, S., Haendiges, J., Keller, E.N., Myers, R., Kim, A., Lombard, J.E., Karns, J.S., Van Kessel, J.S., Haley, B.J. 2018. Genetic diversity of dairy farm – associated Listeria monocytogenes. PLoS One. 13(5):e0197053. https://doi.org/10.1371/journal.pone.0197053.