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:
This is a newly established project that replaces bridging project 8042-32000-099-00D, Ecology and Molecular Epidemiology of Zoonotic Bacterial Pathogens Associated with Dairy Farms. Substantial progress has been made towards the research goals for each of the three objectives. Prevalence and type of resistant E. coli in the feces of BARC dairy animals. In order to establish a baseline of the resistant E. coli types found in the Beltsville dairy herd, sampling was conducted over a several month period from animal groups of different ages. E. coli isolates from each sample group were tested for sensitivity to a panel of 14 antibiotics. Overall, resistance in this population is low, however resistant E. coli were commonly isolated from the newborn calves. (Objective 1) Comparison of the resistomes and metagenomes of veal calves. We sequenced the metagenomes (24 in total) of individual fecal samples from veal calves (3 each) on 4 farms at two different time points (~2 weeks and ~4 months). Analysis of the data is in the early stages. (Objective 1) Establishing cell culture protocols. A significant effort has been made towards establishing the capacity for cell culture in our laboratory and to develop the cell culture protocols needed to conduct the planned research on cell association in Objectives 1 and 2. We began by using an intestinal cell line as a surrogate for the calf or cow intestinal cells; however, we found out that this cell line is contaminated with HeLa Cells and this would cofound the association data. Therefore we switched to using Caco-2 cells and have successfully run preliminary association assays with a few E. coli and Salmonella strains. (Objectives 1 and 3) Ecology of resistant E. coli in dairy herds. We have continued to analyze similarly resistant E. coli isolates from calves and cows on the same farms to compare the similarities between these isolates and to determine if there are resistant strains that persist within a herd. Analysis of pulsed-field gel electrophoresis suggests that, at least some of the strains are highly similar between the animal groups. A selected number of these isolates have been sequenced for further comparisons of their relatedness as well as their resistance-conferring genetic elements. (Objective 1) Comparison of the resistomes and metagenomes of pre-weaned calves and lactating cows. We sequenced the metagenomes of composite fecal samples from adult lactating cows and pre-weaned calves on 17 dairy farms in Pennsylvania. Samples were selected such that an adult cow fecal sample was matched with a pre-weaned calf sample from the same farm. Results indicated that multiple antibiotic resistance genes were present in every sample with tetracycline, aminoglycoside, and macrolide resistance genes the most frequently detected. We also determined that there were more antibiotic resistance genes present in the pre-weaned calf fecal samples than in the adult cow fecal samples and that the pre-weaned calf fecal samples were more similar to each other than they were to adult cow fecal samples. (Objective 1) Genomic analysis of multi-drug resistant (MDR) E. coli strains from pre-weaned calves. We sequenced the genomes of 140 MDR E. coli isolates and investigated their diversity. The majority of strains were from two evolutionary lineages and most strains harbored plasmids. There were four fimH alleles (the major attachment factor of E. coli) that were more frequently detected than other fimH alleles suggesting that these alleles may be involved in attachment to the calf intestinal epithelium. (Objective 2) Comparative genome analysis of Salmonella Dublin from bovine, human, and other sources. Salmonella Dublin is a bovine-adapted serotype and, although S. Dublin is a low prevalence serotype in human infections, hospitalization and mortality rates are higher for Dublin infections vs. more common serotypes. S. Dublin isolates are frequently resistant to > 4 antibiotics. In response to a multi-agency discussion on this pathogen, we assembled a diverse (source, geographical, temporal) set of S. Dublin isolates. The isolates have been sequenced and the sequences will be compared via genomic features of virulence and antimicrobial resistance and whole genome phylogenies will be constructed.
1. Genomic analysis of human foodborne pathogens from dairy cows and other sources. Dairy animals are reservoirs for several zoonotic bacteria species, but humans can also be exposed to these pathogens through other sources. ARS scientists at Beltsville, Maryland, sequenced the genomes of extraintestinal pathogenic E. coli (ExPEC) isolated from dairy calf feces and compared them to closely related human clinical and poultry isolates collected from around the world. Results indicated that ExPEC recovered from dairy calves were closely related to isolates from human intestinal and extraintestinal infections also sequenced the genomes of Salmonella enterica serovar Kentucky recovered from multiple sources in North and South America between 1972 and 2004. This study demonstrated that Salmonella Kentucky isolates from the Americas are more closely related to each other than they are to isolates collected in Europe, South Asia, North Africa, and the Middle East. These data provide further information on the global ecology of extraintestinal pathogenic E. coli and Salmonella Kentucky.
2. Genomic analysis of human foodborne Salmonella Kentucky in Maryland. Salmonella Kentucky is one of the most frequently isolated S. enterica serovars from poultry and dairy cows in the United States but it is an uncommon cause of human salmonellosis; however, human clinical cases are consistently reported each year. To investigate the sources of human salmonellosis caused by S. Kentucky, ARS scientists at Beltsville, Maryland, sequenced the genomes of 16 human isolates collected between 2010 and 2015 and compared them to a database of genomes from the United States and abroad. Results indicated that half of the Salmonella Kentucky infections in Maryland were associated with travel to Northern Africa or South Asia and these strains that were acquired abroad were all found to be resistant to multiple antibiotics including those used to treat Salmonella infections in humans. Additionally, among the human S. Kentucky infections acquired in the United States, multiple cases were associated with the consumption of poultry and the strains isolated from these cases were more closely related to those collected from chickens than from cows and were susceptible to a panel of antibiotics. This work furthers our understanding of the salmonellae that are found in common food producing animals in the United States and those that are imported from abroad.
3. Risk factors associated with antimicrobial resistant E. coli in dairy animals. Animal agriculture is often implicated as a source of antimicrobial resistance and identifying potential management factors that impact prevalence of resistant bacteria in animal feces is important for development of mitigation strategies. Correlations between previous antimicrobial use and resistance in E. coli from the feces of dairy animals were investigated by comparing survey results on previous antimicrobial use and management from 80 dairy farms with the prevalence of resistance in E. coli isolates from manure samples on those farms. Farms reported more antimicrobial use for disease treatment and prevention in calves than in older animal groups, possibly because enteritis and septicemia are much more common in calves. A number of risk factors, including antimicrobial use in each age group, were found to be associated with an increased likelihood of resistance in E. coli, and these associations extended beyond the same antimicrobial classes. This was a small study but supports the need for a better understanding of antimicrobial resistant bacteria in young dairy animals.
Haley, B.J., Kim, S., Pettengill, J., Luo, Y., Karns, J.S., Van Kessel, J.S. 2016. Genomic and evolutionary analysis of two Salmonella enterica serovar Kentucky wequence types isolated from bovine and poultry sources in North America. PLoS One. doi: 10.1371/journal.pone.0161225.