Location: Environmental Microbial & Food Safety Laboratory
2021 Annual Report
Objectives
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.
Approach
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
This is the final report for Project 8042-32000-110-00D, which ended March 16, 2021. New NP108 OSQR approved project 8042-32420-008-00D, entitled “Evaluation of Genetic and Management Factors to Reduce Foodborne Pathogens and Antimicrobial Resistance in Dairy Cattle,” has been established.
In support of Objectives 1 and 2, two studies were conducted collaborating with scientists at Pennsylvania State University. The goal of the first study was to compare the antibiotic resistance (AR) profiles of E. coli shed in the feces of dairy calves that were raised under different management approaches and to characterize changes in the resistant E. coli population from pre-weaning to post-weaning. Feces were collected from 30 calves on each of 13 commercial herds. The AR profiles of ~1000 E. coli isolates from five of the farms that were feeding pasteurized waste milk were characterized, and the metagenomes from a subset of 12 fecal samples were sequenced and analyzed to characterize the microbial community and resistome (AR genes) changes in in the feces of calves from birth through post-weaning. Antimicrobial resistance genes, including those conferring resistance to antibiotics of human health importance, were identified in all samples, but the abundances of these genes were higher in preweaned calves than in postweaned calves. We have also selected 1000 E. coli isolates from pre-and post-weaned calves in 12 herds for comparative genomics analysis. The study is ongoing; however, we anticipate learning critical information on the AR genes, plasmids, sequence types, and virulence factors of resistant E. coli from calves in commercial herds. The second study was a calf experiment conducted to evaluate the impact of feeding waste milk that contains residual antibiotics on resistance in enteric bacteria of calves from birth through post-weaning. Fecal samples were collected up to 2 weeks after weaning (15-18 weeks) from two rounds of 27-30 calves each. E. coli (~4400) isolates were tested for resistance, and 60 fecal samples' metagenomes were sequenced. The microbial community and resistome (AR genes) will be characterized for pre-and post-weaned calves that have been fed milk replacer with or without added antibiotics.
We conducted in vitro experiments to determine if some AR bacteria in calves’ surroundings have a selective advantage over susceptible bacteria in colonizing the calf gut. E. coli growth rates were determined for a diverse set of E. coli isolates in colostrum, and we compared the binding capacity of these isolates to bovine epithelial cells (host-microbe interactions). It appears that AR E. coli do not have an advantage of faster growth rates in colostrum or an enhanced ability to attach to bovine epithelial cells. Our next experiment was designed to determine if non-resistance-conferring genetic attributes in resistant strains may be responsible for a selective advantage in calf gut colonization. We sequenced the genomes of 254 multi-drug resistant (MDR) and pan-susceptible E. coli from lactating cows, dry cows, post-weaned calves, and pre-weaned calves and interrogated the genomes for the presence of 340 virulence-associated genes, plasmid types, and protein-coding genes. Many virulence genes were detected in E. coli from all four groups, and plasmid profiles were highly diverse. Multiple nutrient acquisition and metabolism genes were detected among the MDR-enriched genes, including iron scavenging, myo-inositol transport, and catabolism genes. These data have been used to develop a field study to test the ability of iron levels in colostrum and milk to alter the abundance of MDR E. coli in the calf gut. (Objectives 1 and 2)
In support of Objective 3, Salmonella Kentucky isolates from bovine and human sources were sequenced and compared phylogenetically. Multiple evolutionary lineages, known as sequence types (STs), of S. Kentucky, exist, and these STs are presumed to have significantly different levels of virulence in humans. Most ST152 from cows appear to be genetically distinct from those isolated from poultry and are lacking the major multidrug-resistant plasmid. ST198 isolates are present in dairy cows in the United States but at a relatively low prevalence compared to ST152. Both STs have been isolated from animals and uncooked food products in the U.S . We collaborated with the Maryland Department of Health to sequence all S. Kentucky isolates recovered from human clinical cases in Maryland. These data were mapped to our previous genomic data to determine that in Maryland, most clinical S. Kentucky infections are caused by ST198 and are associated with traveling abroad and that infections are caused by domestically-associated bovine (dairy or beef) and poultry exposures/consumption is rare. We further collaborated with a scientist in Ireland to sequence the genomes of S. Kentucky isolated from food and animal sources in Ireland. Results indicated that very few isolates circulating in agricultural sources in this country are ST198 or ST152, while the majority are ST314. Before this analysis, little was known about this ST, but it appears to be the predominant zoonotic sequence type in Ireland, while the majority of human clinical cases are caused by travel-associated ST314. Results of these studies have helped to understand the ecology and prevalence of S. Kentucky in food sources in the U.S. and abroad and the genomic epidemiology of S. Kentucky infections in the U.S.
Salmonella Dublin is a bovine-adapted serovar and, although it is a low prevalence serovar in human infections, hospitalization, and mortality rates are higher for S. Dublin infections vs. more common serovars. Additionally, S. Dublin isolates are frequently multidrug-resistant. We sequenced the genomes of 115 dairy and beef cattle-associated S. Dublin and analyzed an additional 780 S. Dublin genomes from bovine, human, and other publicly available sources. Results of this study indicated that S. Dublin genomes phylogenetically cluster based on country/continent of isolation, indicating that distinct S. Dublin sub-populations are circulating between host animals within different parts of the world. Within the U.S, there appear to be five major clusters of S. Dublin isolated from bovine animals based on nucleotide sequence divergence, and these clusters do not appear to be related to geography or time of isolation. Many AR genes were identified in these genomes and were in an IncA/C plasmid, indicating that this plasmid is the predominant carrier of AR in S. Dublin in the U.S. Results of these studies help to further our understanding of the genomic ecology of multidrug-resistant S. Dublin from bovine sources in the U.S. and abroad. (Objectives 1 and 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 to interact with cultured bovine cells, we analyzed the attachment and invasion capacities of S. Kentucky strains ST152 that were isolated from different animals within a single herd during an asymptomatic S. Kentucky outbreak and ST198 isolated from a different herd. Isolates that were collected from the herd prior to the outbreak had different binding capacities to those collected during the outbreak; the outbreak most likely outcompeted the pre-outbreak isolates isolates most likely outcompeted the pre-outbreak isolates within the herd. Additionally, we compared the attachment and invasion capacities of strains from 13 Salmonella serovars. S. Dublin was more invasive to bovine epithelial cells than the other serovars. To identify potential factors contributing to differing propensities of cellular invasion, we conducted transcriptomic analyses of S. Dublin and S. Cerro strains when grown in association with bovine epithelial cells. The goal was 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. We identified many differentially expressed genes in the transcriptomes of serovars Dublin and Cerro isolates, indicating that a complex set of factors impact the level of invasiveness. (Objective 3)
We are also analyzing the roles of another potential non-resistance conferring genomic feature, prophage (the genetic material of a lysogenic bacteriophage incorporated into the bacterial genome), on the antimicrobial resistance or sensitivity in their bacterial hosts. We selected an S. enterica lysogen (bacterium infected with lysogenic bacteriophage) that harbored prophage P22 and developed a non-lysogen originating from the wild-type lysogen. Then we constructed a second lysogen by infecting the non-lysogen with bacteriophage P22. We have sequenced the genomes of these isolates and confirmed the presence and absence of prophage P22 in lysogen and non-lysogen strains, respectively. Comparison of physicochemical properties between the lysogens and the non-lysogen has revealed noticeable differences in cellular motility and growth rate. We conducted a substrate utilization and chemical sensitivity assay via phenotypic microarray to identify differences in antimicrobial susceptibility and metabolic potential between the lysogens and the non-lysogen. Initial evaluation of the phenotypic microarray analysis revealed that the lysogens and the non-lysogen had different susceptibilities to multiple antimicrobials, metals, and chemical compounds. Based on this data, we will screen additional antimicrobials and examine the growth kinetics of the lysogens and the non-lysogen in the presence of those antimicrobials to further understand the roles of prophages on AR in bacterial hosts. (Objectives 1 and 3)
Accomplishments
1. Identification of genomic features associated with antimicrobial-resistant E. coli in veal calves. Veal calves remain an underappreciated reservoir of antimicrobial resistance (AMR), and strategies to reduce the carriage of AMR are needed to provide a safer food product to the consumer. To achieve this goal, ARS scientists sequenced the genomes of over 80 E. coli isolates collected from veal calf feces before slaughter and analyzed these data to identify all AMR genes and virulence factors (VFs) therein. Since some VFs are involved in survival within the host, statistical analyses were conducted to determine if VFs are significantly associated with the multidrug-resistant (MDR) trait. These analyses indicated a strong significant association between the MDR trait and iron-scavenging genes and outer membrane proteins. These results are similar to earlier work on E. coli collected from dairy calves and suggest that a potential simple dietary modification (iron supplementation) may reduce AMR in young calves, thereby reducing the prevalence of resistance at the animal and farm level.
2. Global transcriptomes of Salmonella enterica serovars Dublin and Cerro infecting bovine epithelial cells. The impact of S. enterica colonization in cattle is highly variable and often serovar-dependent, but there are significant knowledge gaps in our understanding of genomic and transcriptomic features responsible for the differential interactions between S. enterica serovars with bovine host cells. This study compared the global transcriptomes of the highly pathogenic bovine-adapted S. Dublin and the less pathogenic bovine-adapted S. Cerro during interactions with bovine epithelial cells (host-pathogen interaction) to identify genetic features that impact serovar-related outcomes of S. enterica infections in cattle. Results indicated that the higher invasiveness of the S. Dublin strains to bovine epithelial cells compared with the S. Cerro strains appeared to be a result of a complex set of differentially expressed genes – some genes were upregulated or downregulated in S. Dublin strains. This study furthered our understanding of the potential genomic features of S. enterica that may be responsible for symptomatic or asymptomatic infection/colonization of two bovine-adapted Salmonella serovars in cattle. Results from this study may be used to identify specific drug targets and management strategies that can be leveraged to reduce occurrences of S. enterica serovars in dairy animals.
Review Publications
Taviani, E., Muchongo, A., Kim, S., Van Kessel, J.S., Haley, B.J. 2021. Genomic analysis of antibiotic-resistant and susceptible Escherichia coli isolated from bovine sources in Maputo, Mozambique. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2020.2901.
Salaheen, S., Kim, S., Hovingh, E., Van Kessel, J.S., Haley, B.J. 2020. Metagenomic analysis of the microbial communities and resistomes of feces from veal calves. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.609950.
Kim, S., Van Kessel, J.S., Haley, B.J. 2021. Genome sequences of antibiotic-resistant Escherichia coli isolated from veal valves in the United States. Journal of Global Antimicrobial Resistance. https://doi.org/10.1016/j.jgar.2021.04.024.
