Research Project: Alternatives to Antibiotics and Genomics of Antimicrobial Resistance to Control Foodborne Pathogens in Poultry

Location: Poultry Microbiological Safety and Processing Research Unit

2022 Annual Report

Objectives
1. Identify and determine the presence and contributing factors for antimicrobial resistant foodborne bacteria in poultry and poultry-associated environments. 1.1. Determine the association of antimicrobial resistance (AR) in foodborne bacteria with resistance to biocides, metals, coccidiostats, and ionophores used in poultry husbandry and processing. 1.2. Evaluate the bacterial metagenome of retail poultry. 1.3. Identify and evaluate markers (resistance genes, genetic elements, virulence genes) to define outbreak and persistent foodborne bacteria in poultry. 1.4. Identify antimicrobial resistance gene cassettes (ARCs) and accumulation on plasmids. 2. Identify and evaluate biological and chemical intervention products and alternatives to antimicrobials to control or reduce foodborne pathogens in poultry. 2.1. Develop, validate, and produce multi-subunit vaccines to control Salmonella and Campylobacter in broiler chickens. 2.2. Develop antimicrobial peptides (AMP) as alternatives to antibiotics to reduce foodborne pathogens associated with poultry. 2.3. Identify and develop broad-spectrum bacteriocins to eliminate foodborne pathogens in poultry. 2.4. Utilize phage isolation, whole-genome sequencing (WGS), and metagenomics to identify lytic phage that target Salmonella and pathogenic Escherichia coli.

Approach
Microbial contamination of food products from poultry continues to be a leading cause of foodborne illness. Antibiotics have been used to treat bacterial infections since the mid-twentieth century. Because of their efficacy in treating and preventing disease, antimicrobials have also been widely used in poultry production contributing to antimicrobial resistance (AR) in foodborne pathogens and commensal bacteria. AR among these bacteria has the potential to compromise therapy and remains a global threat to human health. This research project represents a merger of two teams of scientists to provide solutions to colonization of poultry with human pathogens and AR in foodborne pathogens and commensal bacteria from poultry. Two major approaches will be employed: 1) development of alternatives to antibiotics for use in combating foodborne pathogens, and 2) investigations to accurately understand attributes of antimicrobial resistant foodborne pathogens and commensals. Alternatives to antibiotics include vaccines to control foodborne pathogens in live birds while innovative antimicrobial peptides, bacteriocins, and lytic phage will modulate the poultry microbiome to reduce or eliminate colonization by harmful bacteria from poultry to minimize AR and reduce risk to human health. Data generated on resistance to biocides, metals, coccidiostats, and ionophores used in poultry production and processing is a specific concern to the USDA Food Safety and Inspection Service (FSIS). Research designed to determine ecological niches of foodborne bacteria and identify genetic characteristics facilitating transfer of resistance or a fitness advantage will also benefit FSIS. According to FSIS, increased knowledge of the microbial ecology of antimicrobial resistant pathogens on poultry will result in data that the poultry industry can utilize in development of improved pathogen management strategies. Identification of genetic markers which support survival, persistence, and dissemination of foodborne pathogens, especially those that are resistant to antimicrobials, is critical to this research priority. Data and technology from the proposed research will be used to assist other Federal agencies and the poultry and agricultural biotechnology industry in addressing AR in poultry resulting in safer products for the consumer.

Progress Report
ARS researchers in Athens, Georgia, made progress on a subordinate project relating to Sub-objective 1.1 using machine learning to predict co-occurrence of antimicrobial resistance genes in Salmonella and Enterococcus. Causal directed acyclic graphs with Bayesian networks was used to determine co-occurrence of resistance genes in the two bacteria. The dataset consists of approximately 325,000 Salmonella, 15,000 Enterococcus faecium, and 7000 Enterococcus faecalis genomes collected from the National Center for Biotechnology Information website. The AMRFinder plus tool was used to detect antimicrobial, metal, and biocide resistance genes, as well as heat tolerance, and virulence genes. Data was processed using Python to a suitable format for input to perform structural learning using a hill-climbing approach with the help of Bayesian network structure learning (bnlearn.com). The trained Bayesian network models can be used to infer the probability of genes co-occurring given evidence. A prototype Streamlit-based web application was created with a user-friendly interface to infer gene co-occurrence with the help of a trained Bayesian network model. For Sub-objective 1.3, ARS researchers in Athens, Georgia, work on investigating Salmonella serotype Infantis and its plasmid, pESI (plasmid for emerging Salmonella Infantis) was expanded. To address concerns from the poultry industry, an ARS Salmonella Infantis working group was formed by scientists throughout ARS. ARS researchers in Athens, Georgia, hosted a meeting with the National Chicken Council to present the knowledge of the Salmonella Infantis working group and to develop research plans to address this problem. The information was also presented at a meeting of the National Turkey Federation hosted by scientists in Ames, Iowa. Some of the research presented at the meetings included the development of a detection assay which identifies Salmonella Infantis and determines the presence or absence of the pESI plasmid. ARS researchers in Athens, Georgia, also analyzed the Salmonella Infantis chromosome. The study found that Salmonella Infantis with the plasmid have virtually identical chromosomes that are also different from Salmonella Infantis without the plasmid. This indicates that a single clone of Salmonella Infantis acquired the pESI plasmid and then expanded in U.S. poultry. It was also determined that the pESI plasmid could now be found in Salmonella serotypes Infantis, Alachua and Senftenberg isolated from turkeys. Also under Sub-objective 1.3, ARS scientists in Athens, Georgia, conducted a pilot study using “no antibiotic ever” poultry products to standardize methods for sample enrichment and isolation of Salmonella, Escherichia, Campylobacter, Enterococcus, and Staphylococcus. The aim was to utilize one standard buffer to rinse the products that can be used for isolation of each bacterial genus without affecting bacterial viability. Buffered peptone water was better than phosphate buffered saline especially for isolation of Campylobacter. Different combinations of rinsing (parts vs whole product, rinse aliquots vs pelleting aliquots), media and incubation temperature are in the final phase of testing to determine the best methods for isolating each bacterial genus. Antibiotic resistance in Salmonella enterica and Escherichia coli is frequently due to their carrying plasmids that contain antibiotic resistance genes. Under Sub-objective 1.4, the ability of large conjugative plasmids from Salmonella and E. coli to mobilize small kanamycin resistant plasmids was investigated by ARS researchers in Athens, Georgia, and Wyndmoor, Pennsylvania. Eight isolates of Salmonella and E. coli containing large plasmids transferred small kanamycin resistant plasmids. These plasmids were identified and compared to determine characteristics that would allow transfer of the plasmid at different rates. Large plasmids that could not mobilize these small plasmids had disruptions in their transfer genes. These results describe the complexities of small plasmid mobilization and the development of multidrug resistance. The association of small plasmid transfer with large plasmids explains how they may travel together resulting in multidrug resistant pathogens. ARS researchers in Athens, Georgia, made progress on several other projects relating to Objective 1. Seasonal monitoring of surface water as a reservoir and potential vehicle for development of resistant bacteria was suspended due to the pandemic. The citizen scientists from the Upper Oconee Watershed Network could not continue to do the sampling of the 100 locations along the Oconee River. Analysis of the isolates collected from 2015-2020 continued with a project focusing on Salmonella. Salmonella has been traditionally considered a zoonotic foodborne pathogen that is associated with food animals; however, cases associated with contaminated water are on the rise. Isolates (n=1190) from rivers and streams collected between 2015-2017 from a mixed-use watershed in Athens, Georgia, were analyzed. A wide range of serotypes was identified, including those commonly associated with humans and animals, and about half of the isolates were genetically indistinguishable from human clinical isolates. Persistent contamination of surface water with Salmonella that are genetically indistinguishable from clinical isolates supports the role of environmental surface water as a source of Salmonella and possible transmission between the aquatic environment and humans. The National Antimicrobial Resistance Monitoring System (NARMS) investigated adding an environmental component to the surveillance system by including surface water sampling. ARS researchers in Athens, Georgia, worked with other ARS scientists to develop and validate methods for the isolation of Salmonella, E. coli, and Enterococcus from surface water samples. A modification of the Standard Method 9260.B2 developed by scientists in Athens, Georgia, was tested, validated, and selected as the method for the NARMS pilot project. In addition to benefiting NARMS, the Food and Drug Administration, the Environmental Protection Agency, and the Centers for Disease Control and Prevention will also use this method. ARS researchers in Athens, Georgia, conducted continued research on a collaborative extramural-funded project with Mississippi State University, Starkville, Mississippi. The study aims to determine the relationship between antibiotic use and the development of antimicrobial resistance in stocker cattle treated for bovine respiratory disease. Antimicrobial susceptibility and whole genome sequencing of Mannheimia haemolytica, the bovine respiratory pathogen, was completed on 307 isolates from the study. Results of the research will determine if antimicrobial use drives the development of antimicrobial resistance in the pathogens that cause bovine respiratory disease in cattle. ARS researchers in Athens, Georgia, continued analysis of data collected by a visiting Fulbright Scholar from Burkina-Faso. The study investigated Salmonella isolated from slaughtered chickens. Salmonella is an important human pathogen that causes millions of infections world-wide. Because of international travel and global food imports, it is important to study the genetics of antibiotic resistance in Salmonella from countries with no national surveillance system, like Burkina Faso. In this study, 102 Salmonella isolates from slaughtered chickens were whole genome sequenced. About one-third of isolates were multidrug resistant; more than 40% contained mobile genetic element that could allow for increased spread of antibiotic resistance genes. These results demonstrated that chickens are a source of multidrug resistant Salmonella in Burkina Faso and caution should be taken to safely cook and handle chicken meat. Additional research in Burkina-Faso focused on Salmonella from frozen raw fish. Analysis of 28 Salmonella isolates found that they did not carry antimicrobial resistance; however, their serotypes matched those found in local human disease. While imported fish can be an alternative source of protein, frozen products also require proper preparation to prevent foodborne illnesses. Under Sub-objective 2.1, ARS scientists in Athens, Georgia, produced recombinant Salmonella proteins in an E. coli expression system, purified the proteins by affinity chromatography, and tested the proteins for the ability to induce humoral immune response in chickens. The proteins produced various immune responses in the chickens against Salmonella proteins. ARS scientists in Athens, Georgia, conducted other research to investigate the distribution of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequences from Campylobacter jejuni isolates from chicken livers. A total of 155 C. jejuni isolates were detected that harbored the CRISPR sequences, and one type of direct repeat was identified in all 155 isolates. The lengths of the CRISPR loci ranged from 97 to 431 nucleotides. The numbers of the spacers ranged from one to six. Analysis of these spacer sequences indicated that the sequences were grouped into 47 distinctive individual sequences. Further comparison of the spacer sequences with those in databases showed spacer sequence homology to three phage and several plasmids. The results of this research provide important information for development of phage use in the study of bacterial antimicrobial resistance in poultry production.

Accomplishments
1. New quinolone resistance in Campylobacter from chicken livers. Campylobacter is a bacterial pathogen causing human foodborne illnesses worldwide, and outbreaks have been associated with consumption of undercooked chicken livers. Campylobacter from poultry may also be resistant to antibiotics, such as quinolones, used to treat the illness. ARS researchers in Athens, Georgia, assessed antibiotic resistance and analyzed genetic diversity of quinolone resistance in Campylobacter from chicken livers. Isolates were resistant to quinolones, nalidixic acid and ciprofloxacin, and tetracycline. Both common and new mutations in the quinolone resistance determining region were found suggesting that other mechanisms of resistance to the quinolones exist. This data is important for maintaining useful antibiotics for treating Campylobacter foodborne illness as changes in quinolone resistance in Campylobacter may signal future treatment failures.

2. Large conjugative plasmids promote spread of multidrug resistance in pathogens. Salmonella enterica and Shiga-toxin producing Escherichia coli are important human pathogens that are often resistant to antibiotics. This is frequently due to the presence of both large and small plasmids that contain antibiotic resistance genes. Large conjugative plasmids are known to promote the transfer of small kanamycin resistant plasmids, but less is known about the specificity of the transfer. ARS researchers in Athens, Georgia, tested Salmonella and E. coli strains containing large plasmids for their ability to mobilize three different kanamycin resistance plasmids. Two large plasmid types were able to mobilize the tested small plasmids. Large plasmids that could not mobilize the small plasmids had disruptions in their transfer genes. These results describe the complexities of small plasmid mobilization and the development of multidrug resistance. The association of small plasmid transfer with large plasmids explains how they may travel together resulting in multidrug resistant pathogens.

3. The plasmid community of antibiotic resistant enterococci from poultry is diverse and transferable. The plasmid community of antibiotic resistant enterococci from poultry is diverse and transferable. Plasmids are pieces of DNA separate from the chromosome. They may contribute to the problem of antibiotic resistance by transferring resistance genes from commensal bacteria, such as Enterococcus, to pathogenic bacteria. ARS researchers in Athens, Georgia, determined presence and transfer of plasmids and resistance genes from antibiotic resistant enterococci from poultry carcass rinses. Almost half of enterococci were positive for at least one plasmid group. Fourteen plasmid groups were detected, and ten plasmid groups were shared among the enterococci. Eight plasmids were transferred between isolates. Results showed that enterococci from poultry serve as a reservoir of plasmids and antibiotic resistance that can transfer to other bacteria. The presence of this group of enterococci from poultry may contribute to resistance in the human population through contact with poultry products.

4. Surface water plays a hidden role in the cycle of Salmonella infections. Salmonella is traditionally considered a zoonotic foodborne pathogen associated with food animals; however, cases associated with contaminated water are on the rise. The role of surface water as a reservoir and transmission route of Salmonella was addressed in this study by ARS researchers in Athens, Georgia. Seventy percent of the total rivers and streams sampled (n=688) over a three-year period in a mixed-use watershed in Athens, Georgia, were positive for Salmonella. A wide range of Salmonella serotypes was identified, including those commonly associated with humans and animals, and about half of the isolates were genetically indistinguishable from human clinical isolates. Approximately 4.4% of isolates were resistant to antimicrobials and 3.6% were resistant to two or more antimicrobial classes. Persistent contamination of surface water with a high diversity of Salmonella supports the role of environmental surface water as a source of Salmonella and potential public health safety risk.

5. Detection of global antibiotic resistant clones of Staphylococcus aureus in Pakistan. Treatment of Staphylococcus aureus infections has become more complicated due to the emergence of resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA). As a result, the Centers for Disease Control and Prevention has listed antibiotic resistant S. aureus as a serious threat to public health. ARS researchers in Athens, Georgia, collaborated with the University of Haripur, Pakistan, to gather data on global circulation of resistant S. aureus. Clinical S. aureus isolates from Pakistan were tested for resistance to antibiotics. Most of the S. aureus isolated were MRSA and multidrug resistant to drugs commonly used to treat S. aureus infections. MRSA were genetically diverse using molecular typing tests. Regional and global circulating clones implicated in clinical infections were found including clones from the U.S., China, and Argentina. Data from this study are useful to monitor evolution and circulation of resistant bacterial clones and for development of effective infection control measures.

6. Retail food from Egypt contains multiple antibiotic resistant Escherichia coli. Antibiotic resistant bacteria from retail food poses a major public health threat. ARS researchers in Athens, Georgia, and Mansoura University, Egypt, analyzed Escherichia coli from ground beef, chicken carcasses, and humans in Egypt using whole genome sequencing. The analysis revealed the existence of multiple antibiotic resistance genes in the E. coli isolates including colistin resistance genes. The colistin resistance gene in a chicken isolate from this study has also been identified in human and food E. coli isolates from Egypt and China. As E. coli strains containing colistin and other antibiotic resistance genes have only been recently reported, this study shows rapid regional and international spread of these strains among both humans and food animals. Adopting a whole genome sequencing-based surveillance system will facilitate better detection methods and monitoring of international spread of pathogens that are resistant to multiple antibiotics.

7. Improperly handled fish can expose consumers to pathogenic bacteria. Imported commercial fish is a major commodity in Burkina Faso, but fish can be contaminated by pathogenic bacteria that pose a high risk for consumer’s health. ARS researchers in Athens, Georgia, and Burkina Faso analyzed fish sold in open markets in Ouagadougou, Burkina Faso for the presence of Salmonella. Of the 159 fish samples analyzed, 18.9% were contaminated with Salmonella. Six different Salmonella serotypes were found including serotype Tennessee which has been implicated in foodborne outbreaks across the globe. All isolates were susceptible to the tested antibiotics. These results demonstrate that widely consumed imported fish in Burkina Faso are contaminated with Salmonella. Monitoring of imported fish and the conditions where they are sold may decrease potential health risks for consumers especially in low and middle-income countries.

8. Antibiotic resistant Salmonella are prevalent in poultry in Burkina Faso. Salmonella is an important human pathogen that causes millions of infections world-wide. Because of international travel and global food imports, it is important to study the genetics of antibiotic resistance in Salmonella from countries without a national surveillance system, like Burkina Faso. ARS researchers in Athens, Georgia, and Burkina Faso studied the genetics of antibiotic resistance in 102 Salmonella isolates from slaughtered chickens in Burkina Faso. A third of the isolates were multidrug resistant and more than 40% of isolates contained plasmids, a mobile genetic element that could allow for increased spread of antibiotic resistance genes. These results demonstrated that chickens are a source of multidrug resistant Salmonella in Burkina Faso and caution should be taken by producers and consumers to safely handle and cook chicken.

Review Publications
Syed, M.A., Jamil, B., Ramadan, H., Rukan, M., Ali, S., Abassi, S.A., Woodley, T.A., Jackson, C.R. 2021. Genetic diversity of Staphylococcus aureus strains from a tertiary care hospital in Rawalpindi, Pakistan. Microorganisms. https://doi.org/10.3390/microorganisms9112301.
Mcmillan, E.A., Nguyen, L.T., Hiott, L.M., Sharma, P., Jackson, C.R., Frye, J.G., Chen, C. 2021. Genomic comparison of conjugative plasmids from Salmonella enterica and Escherichia coli encoding beta-lactamases and capable of mobilizing kanamycin resistance Col-like plasmids. Microorganisms. https://doi.org/10.3390/microorganisms9112205.
Kagambega, A., Frye, J.G., Belem, S., Mcmillan, E.A., Hiott, L.M., Sharma, P., Gupta, S., Ramadan, H., Soro, D., Barro, N., Jackson, C.R. 2021. Genome analysis of Salmonella strains isolated from imported frozen fish in Burkina Faso. Annals of Microbiology. 71:32. https://doi.org/10.1186/s13213-021-01642-8.
Ramadan, H., Soliman, A.M., Hiott, L.M., Woodley, T.A., Chattaway, M.A., Jenkins, C., Frye, J.G., Jackson, C.R. 2021. Emergence of multidrug-resistant Escherichia coli producing CTX-M-, MCR-1-, and FosA in retail food from Egypt. Frontiers in Cellular and Infection Microbiology. 11: Article e681588. https://doi.org/10.3389/fcimb.2021.681588.
Cho, S., Hiott, L.M., House, S.L., Woodley, T.A., Mcmillan, E.A., Sharma, P., Barrett, J.B., Adams, E.S., Brandenburg, J.M., Hise, K.B., Bateman Mcdonald, J.M., Ottesen, E.A., Lipp, E.K., Jackson, C.R., Frye, J.G. 2022. Characterization of diverse Salmonella isolated from surface water of a mixed-use watershed and their comparison to human clinical isolates. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.00393-22.
Yeh, H., Cox Jr, N.A., Hinton Jr, A., Berrang, M.E., Plumblee Lawrence, J.R., Thompson, T.M. 2021. Prevalence and characterization of quinolone resistance in Campylobacter spp. isolated in chicken livers from retail stores in Georgia, USA. Journal of Food Protection. https://doi.org/10.4315/JFP-21-357.
Gao, Y., Yeh, H., Bowker, B.C., Zhuang, H. 2021. Effects of different antioxidants on quality of meat patties treated with in-package cold plasma. Innovative Food Science and Emerging Technologies. https://doi.org/10.1016/j.ifset.2021.102690.
Oladeinde, A.A., Abdo, Z., Press, M.O., Cook, K.L., Cox Jr, N.A., Zwirtzitz, B., Woyda, R., Lakin, S.M., Thomas Iv, J.C., Looft, T.P., Cosby, D.E., Hinton Jr, A., Guard, J.Y., Line, J.E., Rothrock Jr, M.J., Berrang, M.E., Herrington, K., Zock, G.S., Plumblee Lawrence, J.R., Cudnik, D., House, S.L., Ingram, K.D., Lariscy, L., Wagner, R., Aggrey, S.E., Chai, L., Ritz, C. 2021. Horizontal gene transfer is the main driver of antimicrobial resistance in broiler chicks infected with Salmonella enterica Serovar Heidelberg. mSystems. 6(4):e00729-21. https://doi.org/10.1128/mSystems.00729-21.
Mcmillan, E.A., Hiott, L.M., Jackson, C.R., Frye, J.G., Kagambega, A., Ramadan, H., Bouda, C., Soro, D., Sharma, P., Gupta, S., Barro, N. 2022. Resistance genes, plasmids, multilocus sequence typing (MLST) and phenotypic resistance of Salmonella isolated from slaughtered chickens in Burkina Faso. Antibiotics. https://doi.org/10.3390/antibiotics11060782.
Jackson, C.R., Cho, S., Mcmillan, E.A., Barrett, J.B., Hiott, L.M., Woodley, T.A., House, S.L., Frye, J.G. 2022. Distribution and transfer of plasmid replicon families among multidrug resistant Enterococcus faecalis and Enterococcus faecium from poultry. Microorganisms. https://doi.org/10.3390/microorganisms10061244.
Yeh, H., Awad, A., Rothrock Jr, M.J. 2021. Detection of Campylobacter jejuni diversity by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from poultry sources. Veterinary Medicine and Science. https://doi.org/10.1002/vms3.622.