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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Environmental Microbial & Food Safety Laboratory » Research » Research Project #440225

Research Project: Evaluation of Genetic and Management Factors to Reduce Foodborne Pathogens and Antimicrobial Resistance in Dairy Cattle

Location: Environmental Microbial & Food Safety Laboratory

2022 Annual Report

Objective 1: Elucidate the role of accessory genetic elements including siderophores, metabolism genes and transport factors on the persistence of multi-drug resistant bacteria in dairy animals and their environments. Sub-objective 1.A: Compare the growth of MDR and susceptible E. coli and S. enterica strains encoding accessory siderophores and iron transport mechanisms in bovine feces with and without iron supplementation. Sub-objective 1.B: Evaluate the role of dietary iron supplementation in the first two weeks after birth on MDR E. coli abundance in the calf gut. Sub-objective 1.C: Identify the effects of accessory myo-inositol transport and metabolism genes on the growth of MDR E. coli and susceptible E. coli strains. Objective 2: Evaluate the feasibility of commensal bacteria as modulators of pathogenic Salmonella enterica and antibiotic resistant E. coli carriage. Sub-objective 2.A: Examine the ability of commensal bovine gut bacteria and non-pathogenic S. enterica to outcompete pathogenic S. enterica strains in the bovine gut environment. Sub-objective 2.B: Examine the ability of susceptible E. coli strains to outcompete MDR E. coli strains in the gut of newborn calves. Objective 3: Identify the management factors involved in persistence of multi-drug resistant bacteria in milk-fed dairy calves.

Dairy animals, including milk-fed veal, and their farm environments are reservoirs for zoonotic pathogens and antimicrobial resistance and the impetus for this project is to develop solutions for reducing the prevalence of resistant and human pathogenic bacteria harbored by these animals. Our previous work identified preweaned calves as an important reservoir for antibiotic resistance; resistant bacteria appear to outcompete sensitive bacteria in the very young calf gut. We identified accessory genetic elements associated with the acquisition, transport, and metabolism of iron and myo-inositol, two essential nutrients for calf development, that may enhance the ability of resistant Enterobacteriaceae to outcompete sensitive strains in the neonatal calf gut. We will investigate the impact of these accessory genes on the ability of resistant strains to outcompete sensitive bacteria in iron-replete growth conditions using in vitro and in vivo approaches, as well as the impact of myo-inositol on resistant strain selection in vitro. We will also explore the ability of bovine commensal bacteria that are hypothesized antagonists of Salmonella to modulate the abundance of pathogenic S. enterica via competitive in vitro growth assays and by evaluating the ability of these commensals to prevent pathogenic S. enterica from attaching to and invading bovine epithelial cells. Similarly, animal studies will be conducted to evaluate the ability of antibiotic-susceptible E. coli to outcompete resistant E. coli in newborn calves with the aim of reducing the carriage of resistance in preweaned calves. Finally, we will conduct on-farm studies to identify management factors that influence the abundance and types of resistance harbored by veal calves. The ultimate goal of the project is to develop novel and practical mitigation approaches that can be employed by the dairy industry.

Progress Report
This is the first annual report for the Project 8042-32420-008-000D entitled “Evaluation of Genetic and Management Factors to Reduce Foodborne Pathogens and Antimicrobial Resistance in Dairy Cattle”, which began March 17, 2021. In support of Objectives 1 and 2, two studies were conducted. The goals of these two studies were to determine the role of iron in the presence and persistence of multidrug resistant (MDR) Escherichia coli in the dairy calf gut. For the first study, twenty-nine E. coli isolates from dairy animals were selected to represent variably encoding resistance genes and iron-scavenging systems. These isolates were grown in three different conditions of iron availability and their growth rates were observed. Growth conditions included media with iron added, media with iron depleted, and unaltered conventional growth media (Luria-Bertani broth). The aim of this study is to determine if, under tightly controlled conditions, iron availability is associated with enhanced growth in E. coli that encode iron-scavenging genes, regardless of the carriage of resistance genes. The second study was a controlled calf experiment with collaborators at the Pennsylvania State University that was conducted to evaluate the impact of supplementing milk replacer with iron on the presence and abundance of multidrug-resistant E. coli and total resistance genes in the preweaned calf gut. For this experiment, calves were acquired from commercial dairy facilities and separated into two groups that were fed iron-supplemented milk replacer or milk replacer that was not supplemented with iron. Fecal samples were collected daily for seven days and then weekly until the calves were 6 weeks old. E. coli was isolated from the samples and a portion of the feces was preserved for downstream shotgun metagenomics. Fecal and serum iron levels will be analyzed to determine the amounts of iron absorbed and excreted from each animal. Currently, the E. coli isolates are being analyzed for antimicrobial resistance phenotypes and these data will be used to identify isolates for whole genome sequencing and preserved fecal samples for shotgun metagenomic analysis. We are also about to start analyzing the preserved fecal samples by quantitative PCR (qPCR) to determine the presence and abundance of specific antimicrobial resistance genes and to determine if iron supplementation influenced the dynamics of these genes in the calf gut. In support of Objectives 1 and 3, with the help of collaborators at Pennsylvania State University, we have collected intestinal samples of in utero calves whose dams were slaughtered. Currently, it is believed that calves become colonized with antimicrobial-resistant bacteria from the dam during birth and their immediate environment in the beginning days of life. However, this has yet to be proven. Evidence from human studies suggests that some prenatal gut colonization may occur in utero by horizontal transmission from the mother to offspring. For this study we aim to evaluate the stage at which initial colonization of the calf gut by antimicrobial-resistant bacteria occurs. Preliminary analyses using culturing techniques has shown that the in utero calf gut contents do not harbor bacteria that are able to grow on Escherichia coli and Enterobacteriaceae-selective media, which suggests that the transfer of Enterobacteriaceae does not occur from dam to calf in utero. The next step in this analysis is to conduct shotgun metagenomic sequencing on these intestinal contents. These data will be analyzed to identify which (if any) taxa are present in the in utero calf gut, and what antimicrobial, biocide, and metal resistance genes they may carry as well as genes that may be involved in any significant pathway involved in bacterial survival in utero. Metagenomic analysis of the sequencing reagents and exterior washes of the materials used in the transport and processing of the samples will also be conducted to identify any potential contaminants that may interfere with our analysis. These samples are considered low biomass samples and therefore contain very small amounts of DNA and care must be taken to account for any potential contaminant DNA, which will need to be removed from the data in silico. We expect this project to be completed during the Fall of 2022. In support of Objective 3, we are currently conducting a collaborative study with researchers at University of Vermont, looking at the prevalence of antimicrobial resistance on farmstead and artisan cheese farms in Vermont. For this project samples will be collected from the dairy environment, including feed and water, as well as from the lactating cows, bulk tank milk, pasteurized milk, and final cheese product. The samples will be analyzed by shotgun metagenomics, quantitative PCR (qPCR), and traditional culturing methods. Management practices will also be collected during the study. The aims of this study are to identify the presence of antimicrobial resistance genes in farmstead and artisan cheese farms, the sources of these resistance genes, and the on-farm practices that influence their dynamics. A post-doctoral researcher, stationed at the University of Vermont, has recently been hired to work on this project and is currently learning metagenomics analysis methods as well as preparing to begin sample collection and processing. In support of Objective 1 we have sequenced the genomes of 1000 non-redundant E. coli isolates collected from 12 commercial dairy herds in Pennsylvania. Approximately half of these were collected from preweaned dairy calves and half were collected from the same animals after the weaning transition was completed. The objective of this study is to identify genomic features (e.g., accessory genetic elements, metabolic genes and transport factors) that may be associated with the disproportionately large population of antimicrobial-resistant E. coli that have been observed in pre-weaned dairy calves. Preliminary results revealed that E. coli population structures (sequence types) dynamically shift from pre- to post-weaning in calves; some sequence types are mostly found in pre-weaned calves, while some are mostly found in post-weaned calves. We further identified the virulence factors of these isolates and observed that the Shiga toxin gene-harboring E. coli (possible STEC) were often resistant to antimicrobials and were more abundant in post-weaned calves compared with calves that have not yet been weaned. Using a comparative genomic analysis, we have identified potential genes that have a stronger association with AMR E. coli than with susceptible E. coli. We are currently exploring those genes and the associated pathways for possible links with AMR E. coli carriage in calves.


2. Identification of E. coli isolates from dairy animals in the United States encoding Fosfomycin-resistance genes. Fosfomycin is considered a reserve-group antibiotic (also known as a ‘last-resort antibiotic’) that has been used to successfully treat people for infections caused by multidrug-resistant bacteria. ARS researchers at Beltsville, Maryland, analyzed the genomes of more than 300 Escherichia coli from dairy animals in Pennsylvania and identified Fosfomycin resistance genes in a small subset (< 2%) of the isolates. Fosfomycin-resistance genes were also identified in 117 E. coli genomes from other bovine sources and products in the U.S., 23 of which were isolated from dairy cows in 11 states. Further analysis indicated that there was a very low level of genomic diversity among the Fosfomycin-resistant E. coli. The identification of very low levels of resistance to this reserve-group antibiotic in the bovine gut suggests that monitoring should be implemented for potential enrichment of this resistant phenotype.

3. Transcriptome variability of two dairy-associated Salmonella enterica serovar Dublin strains during interactions with bovine epithelial cells. Salmonella enterica serovar Dublin (S. Dublin) is a human and animal pathogen that can cause severe illness in humans and cattle. S. Dublin is a genetically diverse serovar and currently there is little information regarding the impact of this diversity on colonization of the bovine gut. ARS researchers at Beltsville, Maryland, investigated the host-pathogen interactions of two, genetically different, S. Dublin strains and demonstrated that strain-dependent gene expression profiles have a role during S. Dublin infection of bovine epithelial cells. However, despite the potential for different molecular mechanisms of host-pathogen interactions, many genetically-distinct S. Dublin strains may have similar infection and survival rates within bovine epithelial cells. This study furthered our understanding of the genes that are important during infection/colonization of S. Dublin in cattle.

Review Publications
Haley, B.J., Kim, S., Salaheen, S., Hovingh, E., Van Kessel, J.S. 2022. Virulome and genome analyses identifies associations between antimicrobial resistance genes and virulence factors in highly drug-resistant Escherichia coli isolated from veal calves. PLoS ONE. 17(3):0265445.
Kim, S., Van Kessel, J.S., Haley, B.J. 2022. Draft genome sequence of an Escherichia coli ST38 strain isolated from dry cow feces on a commercial dairy operation. Microbiology Resource Announcements.
Soltys, R.C., Sakomoto, C., Oltean, H.N., Guard, J.Y., Haley, B.J., Shah, D.H. 2021. High-resolution comparative genomics of Salmonella Kentucky aids source tracing and detection of ST198 and ST152 lineage-specific mutations. Frontiers in Sustainable Food Systems. 5:6953368.
Slowey, R., Kim, S., Prendergast, D., Madigan, G., Van Kessel, J.S., Haley, B.J. 2021. Genomic diversity and resistome profiles of Salmonella enterica subsp. enterica serovar Kentucky isolated from food and animal sources in Ireland. Zoonoses and Public Health. (10):1011.
Sapountzis, P., Teseo, S., Otani, S., Aarestrup, F., Forano, E., Suen, G., Tsiamis, G., Van Kessel, J.S., Haley, B.J., Huws, S. 2022. FI: The fecobiome initiative. Foodborne Pathogens and Disease. 19(17):441-447.