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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

2023 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 second annual report for the project and significant progress has been made in support of Objectives 1 and 2. In support of Objective 1.A and 2.A, we have worked towards establishing high-throughput protocols for comparing the growth rates of multidrug resistant (MDR) and susceptible E. coli and Salmonella using 96-well plate readers. The variability in results due to background noise is too great to accurately detect differences between strains using this method. We are reducing our strain selection and changing our approach to accommodate this result. In support of Objective 1.B, we have analyzed fecal samples and bacterial isolates from a calf study that we conducted last year. The study was designed to look at the potential impact of iron supplementation in the diet of very young dairy calves on colonization of the calf gut by antimicrobial-resistant E. coli. Over 1700 E. coli isolates were characterized for their antibiotic sensitivity. Additionally, the metagenomes of 50 fecal samples were sequenced. Analysis of the phenotypic and metagenomic data has been initiated. In support of Objective 2.A, we have evaluated the inhibitory potentials of commensal S. Cerro and Kentucky to outcompete pathogenic S. enterica serotypes including Newport, Dublin, Montevideo, Typhimurium, and Heidelberg. We conducted growth assays designed to detect inhibitory effects of secreted extracellular chemicals in the supernatants from S. Cerro and Kentucky overnight cultures on growth of the pathogenic serotypes. These experiments are on-going but initial results indicate minimal, if any, inhibitions. In support of Objective 2.B, we have conducted a pilot competitive exclusion study with collaborators at Penn State University in University Park, Pennsylvania. We determined that a pilot was needed to provide preliminary data that could be used to design a full animal study that would require significant resources. We inoculated each of 4 calves within 24h of birth with single or combinations of E. coli strains to determine the ability of certain susceptible strains to outcompete multi-drug resistant strains in the neonatal calf gut. Fecal samples were collected from the calves for 14 days and E. coli were isolated from each sample. PCR assays were developed to detect the inoculated strains and the E. coli isolates were tested to determine if the inoculated strains persisted in the calf gut. We are also sequencing some of the isolates to further characterize the persistent E. coli population. Results from this pilot study will inform us of how best to approach a full calf trial. In support of Objective 1, we completed analyzing the genomes of >1000 E. coli that were isolated from the feces of dairy calves once before weaning and once after weaning. The genomes were characterized for their relatedness, antimicrobial resistance gene content, virulence gene content, plasmid replicons and sequence type assignment. We looked for trends in these characteristics to see if there were differences between strains isolated before weaning and those isolated after weaning. In response to observations by scientists at FSIS, we conducted a study on a human salmonellosis outbreak caused by a pansusceptible strain of Salmonella Newport. This serotype is highly pathogenic for both humans and cows, but infections caused by this pansusceptible strain resulted in a significantly higher hospitalization rate than typical S. Newport infections. Genome sequences from these outbreak strains and closely related genomes were downloaded from NCBI and were analyzed for their relatedness, presence of virulence genes, antimicrobial resistance genes, and plasmid replicons.

1. Feeding waste milk to dairy calves can lead to greater antimicrobial resistance in their feces. Feeding waste milk, predominantly milk that cannot be marketed to humans, to calves is a common practice in the U.S. dairies and waste milk often contains antibiotic residues. ARS scientists in Beltsville, Maryland, and Pennsylvania State University jointly characterized antimicrobial resistance in feces from preweaning and postweaning calves on dairy farms that fed waste milk to their calves, and also conducted a controlled study where calves were fed milk with or without added antibiotics and weaned at two different ages. Based on characterization of the antimicrobial resistance of Escherichia coli, an enteric bacteria present in the gut of humans and warm-blooded animals, and the resistance genes in the total microbial population of the calf fecal samples, there was a high level of antimicrobial resistance in the calves during the preweaning phase of life and this was followed by a decline in resistance in the immediate postweaning period. The presence of antibiotic residues in the diet of calves in the controlled study led to higher levels of resistance in the fecal bacteria and this was not impacted by age at weaning. These results improve understanding of the potential impact of feeding waste milk on antimicrobial resistance and identification of the critical times during calf management for mitigating levels of resistance.

2. Prior to weaning, dairy calves are reservoirs of specific suites of highly multidrug-resistant pathogenic Escherichia coli. Fecal waste from cows at all stages of development can potentially carry pathogenic Escherichia coli. ARS scientists in Beltsville, Maryland, and Pennsylvania State University, University Park, Pennsylvania, isolated and analyzed E. coli from the feces of calves on dairy farms prior to weaning and again after weaning. Based on an analysis of the sequence types (strain types) and virulence genes in the genome sequences of these E. coli, it was determined that preweaned calves are more likely to harbor the pathogenic sequence types 69 and 117 (ST69 and ST117) that are known to cause severe illness in the U.S. and abroad. Further, this analysis demonstrated that the strains carried by calves prior to weaning were different than those carried after weaning, indicating that the E. coli population changes as calves get older and their diets change. These results help to understand how animal age may impact their potential to carry certain antimicrobial-resistant bacteria and will allow for the development of management-related interventions to reduce this carriage.

3. Acquisition of antimicrobial-resistant bacteria by calves most likely occurs after birth, not in utero. Calves can carry antimicrobial-resistant bacteria in the gastrointestinal tracts during their first days of life, but the sources of these bacteria and the timing of this colonization have not been well-elucidated. The possibility of in utero transmission from dam to calf has not been evaluated as a possible early-life seeding mechanism of the calf gut. To evaluate if this occurs, scientists from ARS Beltsville, Maryland, and University Park, Pennsylvania, tested the intestinal contents of prenatal calves for the presence of bacteria and antimicrobial resistance genes using metagenomic sequencing. Results of this study indicated that in utero transmission of antimicrobial-resistant bacteria does not occur and that colonization of the calf gut by these bacteria happens very soon after birth or during birthing process. This study provides a better understanding of when transmission of antimicrobial-resistant bacteria to the calf gut occurs, and this information can be used to identify mitigation strategies.

4. A pansusceptible Salmonella Newport that caused an outbreak associated with increased hospitalizations harbored a unique plasmid. During an ongoing multiyear Salmonella Newport outbreak associated with travel to Mexico and consumption of soft cheeses and beef, a cluster of infections resulting in a significantly increased hospitalization rate was observed. Scientists from the Food Safety Inspection Service (FSIS) sequenced the genomes of isolates from this outbreak and ARS scientists in Beltsville, Maryland, analyzed these genome sequences to find differences between the genomes of majority of the outbreak strains and the strains associated with increased hospitalization rates. The latter strains had lost a plasmid that encodes multiple antimicrobial resistance genes and gained a small plasmid that is infrequently found in other strains and has similarities with other plasmids known to result in enhanced colonization of the host. Results of this study help to understand the dynamics of large Salmonella outbreaks and the genetic elements that may result in different health outcomes.

Review Publications
Salaheen, S., Kim, S., Springer, H., Hovingh, E., Van Kessel, J.S., Haley, B.J. 2023. Genomic diversity of antimicrobial-resistant and Shiga toxin gene-harboring non-O157 Escherichia coli from dairy calves. Journal of Global Antimicrobial Resistance. 33:164-170.
Salaheen, S., Kim, S., Van Kessel, J.S., Haley, B.J. 2022. Differences between the global transcriptomes of Salmonella enterica serovars Dublin and Cerro infecting bovine epithelial cells. BMC Genomics.
Salaheen, S., Kim, S., Springer, H., Hovingh, E., Van Kessel, J.S., Haley, B.J. 2023. Characterization of antimicrobial resistance genes and virulence factors in the genomes of Escherichia coli ST69 isolates from preweaned dairy calves and their phylogenetic relationship with poultry and human clinical strains. Microbial Drug Resistance.
Salaheen, S., Kim, S., Van Kessel, J.S., Haley, B.J. 2022. Differences between the global transcriptomes of Salmonella enterica serovars Dublin and Cerro during infection of bovine epithelial cells. BMC Genomics.
Gorji, H., Van Kessel, J.S., Haley, B.J., Husarik, K., Sonnier, J.L., Shahabi, S., Zadeh, H., Chan, D.E., Qin, J., Baek, I., Kim, M.S., Akhbardeh, A., Sohrabi, M., Kerge, B., Mckinnon, N., Vasefi, F., Tavakolian, K. 2022. Deep learning and multiwavelength fluorescence imaging for cleanliness assessment and disinfection in food services. Frontiers in Remote Sensing.