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

Location: Poultry Microbiological Safety and Processing Research Unit

2023 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
The effect of using different biocides sequentially to kill Salmonella Infantis was determined under Sub-objective 1.1. Para-acetic acid, sodium hypochlorite, and calcium hypochlorite were tested. To evaluate the use of biocides in sequential combination, 96 well plates were made with 2-fold serial dilutions with the first compound to be tested. After inoculation with Salmonella Infantis carrying the pESI (plasmid for Emerging Salmonella Infantis) multidrug resistant/virulence plasmid, additional chemicals were added at 15 second intervals, followed by neutralizing buffered peptone water. After treatment, bacterial cells were enumerated by spreading on blood agar plates. Results determined the combination and order of application of the biocides that yielded the highest level of killing Salmonella Infantis with pESI. Work continued on machine learning to predict co-occurrence of antimicrobial resistance in Salmonella and Enterococcus relating to Sub-objective 1.1. Progress was made to generate a causal directed acyclic graph from a Bayesian network to determine the co-occurrence of antibiotic resistance phenotypes of Salmonella. Bayesian parameters were used to estimate and evaluate values of the conditional probability distributions of the Salmonella network. The dataset consists of approximately 4,471 Salmonella isolates and 5,992 Enterococcus isolates from retrospective antimicrobial susceptibility testing surveillance datasets collected by the Food and Drug Administration. Resistance phenotype datasets were preprocessed using Python before network generation using the Bayesian network structure learning Python software package. Bacterial metagenomes of retail poultry were analyzed under Sub-objective 1.2. DNA was extracted from whole product rinsates from conventional and “no antibiotic ever” retail poultry products (n=34). Four kits were evaluated to determine which kit worked best to improve the purity of extracted DNA. Shotgun metagenomic sequencing for preliminary samples (n=8) was done followed by metagenomic analysis with assembly and taxonomic analysis. Under Sub-objective 1.3, conventional (n=40) and “no antibiotic ever” (n=40) retail poultry products collected quarterly from September 2022 to May 2023 for sub-objective 1.2 were also used to isolate outbreak strains of Salmonella and persistent strains of Campylobacter, Escherichia, Enterococcus, and Staphylococcus. Conventional samples were positive for Salmonella (35%), Campylobacter (2.5%), Escherichia coli (97.5%), Enterococcus (97.5%), and Staphylococcus (52.5%). “No antibiotic ever” products were positive for lower levels of Salmonella (32.5%), E. coli (87.5%), Enterococcus (95%), and Staphylococcus (32.5%), but higher for Campylobacter (17.5%). No Escherichia albertii were recovered from any of the poultry products. Salmonella serogrouped into groups B, C1, C2, C3, D1, and E. Both Campylobacter coli and C. jejuni were identified among the Campylobacter isolates. Work by the ARS Salmonella Infantis Working Group continued under Sub-objective 1.3. The working group was formed in collaboration with the Office of National Programs to address the threat of Salmonella serotype Infantis and pESI to U.S. poultry. The group met monthly to present and discuss research related to controlling Salmonella in poultry and to present this data to other federal agencies and stakeholders. A strategic planning meeting was established with the Office of National Programs, the National Chicken Council, and the National Turkey Federation to develop a response to mitigate this threat. Genomic analysis of Salmonella Infantis and the Food Safety and Inspection Service whole-genome sequencing data determined that Infantis with pESI was responsible for the increase of Infantis in chickens and turkeys, and that expansion of this strain is responsible for a continuing outbreak of human illness. The analysis also showed that pESI has transferred to new Salmonella serotypes in turkeys, including Senftenberg. An assay was developed for the Food Safety and Inspection Service for detection of Salmonella Infantis and pESI under Sub-objective 1.3. Using whole-genome sequencing data and bioinformatic analyses, gene targets were first identified. A PCR assay was developed from the identified targets to detect the unique clustered regularly interspaced short palindromic repeats (CRISPR) sequence in the Infantis chromosome and three genes found exclusively in pESI. Analysis of nearly 200 Salmonella isolates confirmed that the PCR assay could accurately detect Salmonella Infantis and pESI. In collaboration with scientists from a commercial company, projected accuracy and specificity of the PCR assays were determined using bioinformatic analysis of over 30,000 Salmonella sequences in the public database. This analysis showed that the CRISPR target was more than 95% accurate and that the pESI PCR assays that detect the unique repA gene from pESI was greater than 99% accurate. A real-time detection assay is being developed for these two genes to meet the Food Safety and Inspection Service requirements. For Sub-objective 2.1, recombinant Salmonella sub-unit proteins were tested in chickens to determine if these proteins could induce humoral immune responses in hosts. No detrimental signs were observed in chickens from the immunized and un-immunized groups during the six-week experimental period. Three isotypes of broiler immunoglobulins against the recombinant proteins were detected and quantification was done using automated capillary immunoblot assays. Immunoglobulin G antibody response from immunized chickens was higher than that from the un-immunized group both two- and three-weeks post immunization. Immunoglobulin M antibody from the immunized group was higher than that from the un-immunized chickens two weeks post-immunization, but immunoglobulin M declined rapidly in the immunized chickens between two to three weeks post-immunization. Immunoglobulin A antibody was also detected against proteins in the immunized group, higher than in the un-immunized chickens. Immunoglobulin A antibody declined in the immunized chickens between two to three weeks post-immunization. Under Sub-objective 2.1, a collaboration with Emory University, Atlanta, Georgia, also continued to develop and produce an mRNA-based vaccine against Salmonella Infantis for use in poultry. For development of antimicrobial peptides in Sub-objective 2.2, expression of the RL-37 antimicrobial peptide in the E. coli thioredoxin fusion model required using an enterokinase cleavage recognition site between each monomer. Digestion with enterokinase to release the 4.1 kDa RL-37 was cost prohibitive as the enterokinase is expensive. Collaborators in Peoria, Illinois, were redirected to attempt expression of a modified version of RL-37 in an alternate yeast vector, Pichia pastoris, and in Bacillus subtilis, a probiotic strain, with addition of the signal and pro-region sequences. This will increase stability in yeast allowing for cost-effective expression and recovery and direct feeding of a probiotic strain to protect RL-37 for delivery to the intestines. A quote for the yeast modified construct has been received from a commercial company. A second antimicrobial peptide, also determined to have in vitro inhibitory effects on Salmonella and Campylobacter target strains, was synthesized with modifications to enhance lipopolysaccharide binding potential. This antimicrobial peptide was evaluated in vitro to determine if this lipopolysaccharide targeting fusion peptide had increased activity compared to the unmodified peptide. No modification benefit was indicated. Efforts to evaluate directly synthesized RL-37 in vivo were begun. Under Sub-objective 2.3, work began on identification of bacteriocins using genome mining of whole genome sequences from multidrug resistant Enterococcus isolates from poultry. Genomes were screened using the program antiSMASH which provides rapid genome-wide identification, annotation, and analysis of secondary metabolite biosynthesis gene clusters in bacterial genomes. Of the 30 genomes analyzed, six biosynthetic gene clusters were identified and the presence of known bacteriocins (enterocin A, pantocin, and microcin) were predicted. For Sub-objective 2.4, work began on development of Bdellovibrio as a probiotic against Salmonella Infantis, complementary to the development of bacteriophage to reduce levels of Salmonella. Bdellovibrio are a predatory bacteria known for their ability to kill Gram-negative bacteria, suggesting it could be a used to reduce the levels of Salmonella. Three established Bdellovibrio strains were tested in a predation assay on 10 Salmonella strains, with different serotypes and antimicrobial resistances including S. Infantis with and without the pESI plasmid. Bdellovibrio strains were able to kill all Salmonella tested. Preliminary reports support the ability of the Bdellovibrio strains to prey on Salmonella. Soil samples were collected from the U.S. National Poultry Research Center campus and tested for the presence of predatory bacteria. Plate assays using Salmonella Infantis with pESI as prey yielded plaques indicating the activity of predatory bacteria. Some of the plate assays also had plaques with morphology indicating the presence of bacteriophage. The Bdellovibrio and bacteriophage have been isolated for further evaluation and a method to isolate both predatory bacteria and bacteriophage in the same assay has been developed to screen additional samples.

Accomplishments
1. Buffered peptone water formulation does not influence growth of pESI-positive Salmonella Infantis. Salmonella is an important human bacterial pathogen that causes foodborne outbreaks. There are more than 2,500 serotypes of Salmonella and over the years different serotypes have been prevalent in poultry and human disease outbreaks. However, Salmonella serotype Infantis has been rarely detected in poultry. Starting in 2016, S. Infantis has been more frequently detected in chickens and turkeys. While S. Infantis detection has increased, the method used by USDA to determine the Salmonella status of poultry changed to include a new Salmonella detection broth designed to counteract residual antimicrobial processing chemicals and allow more accurate detection of Salmonella. The co-incident nature of the increase in detection of S. Infantis and use of a new broth for detection led to speculation that the new broth may be promoting the growth and detection of S. Infantis at a higher rate than the original broth. ARS researchers in Athens, Georgia, compared survival during overnight cold shipment and growth during incubation of S. Infantis poultry isolates in the new and original broth. No differences in the two broths were found suggesting that the change in regulatory methodology did not cause the increase in S. Infantis detection from poultry meat. These results are important to improve poultry production and processing to assure that a safe and wholesome product is delivered to consumers.

2. Increased occurrence of Salmonella Infantis in poultry due to plasmid-encoded genes. Salmonella Infantis has become prevalent in U.S. poultry and is also causing an outbreak of human infections. The virulence plasmid associated with this strain has been identified as being responsible for this phenomenon, but very little is known about the chromosome of the Infantis that carries this plasmid. ARS researchers in Athens, Georgia, completed a bioinformatic study of over 3,200 Salmonella lnfantis genomes to understand differences in the chromosomes between groups that did and did not carry the plasmid. The chromosomes of the group that carry the plasmids are far less diverse than the non-plasmid group indicating that plasmid carriage resulted from a single plasmid acquisition event rather than several events. Further investigations showed there are 58 single nucleotide changes present in the group with the plasmid, none of which are in Infantis without the plasmid. Additionally, no specific genes were present in just one group. This work has helped to better understand the genetics of Salmonella lnfantis and will inform future interventions for the reduction of it in poultry products.

3. Identification and quantification of chicken immunoglobin in sera by automated capillary immunoassays. The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. To reduce these potential problems, ARS researchers in Athens, Georgia, applied a capillary electrophoresis-based immunoblot system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella proteins. The results showed that a single band of each protein was detected in the gel-like images by this system after purification by chromatography. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

4. Antimicrobial peptides are effective in killing Campylobacter. Campylobacter is a major cause of acute human diarrheal illness. Broiler chickens constitute a primary reservoir for Campylobacter jejuni leading to human infection. Consequently, there is a need for developing novel intervention methods such as antimicrobial peptides, small proteins which have evolved in most lifeforms to provide defense against microbial infections. To date, over 3000 antimicrobial peptides have been discovered; however, few of them have been analyzed specifically for ability to kill campylobacters. ARS researchers in Athens, Georgia, selected and evaluated a set of 11 unique chemically synthesized antimicrobial peptides for ability to inhibit growth of C. jejuni. Six of the antimicrobial peptides tested produced zones of inhibition against C. jejuni. These antimicrobial peptides are attractive subjects for future study and potential in vivo delivery to poultry to reduce Campylobacter populations.

5. Antimicrobial resistance genes in the environment are linked to aging wastewater infrastructure. Antibiotic resistance is a threat to human health. How the genes that cause antimicrobial resistance are transferred in the environment is poorly understood. Agricultural runoff and human waste are known sources of antimicrobial resistance genes in aquatic systems. ARS researchers in Athens, Georgia, collaborated with the University of Georgia, Athens, Georgia, to quantify genetic markers of fecal source genes and antimicrobial resistance genes in water samples collected each season over a 5-year period from sites in the Upper Oconee watershed (Georgia, USA). This area is characterized by agricultural and urban development. Widespread fecal contamination was found from human, ruminant, and poultry sources. In addition, 73% of samples tested positive for at least one of the six targeted resistance genes. Antimicrobial resistance genes were strongly correlated with human fecal genes; however, many highly contaminated samples were not associated with sewage outfalls, an expected source of fecal and resistance gene pollution. Antimicrobial resistance gene and fecal marker data was combined with geospatial data on land use/land cover and wastewater infrastructure across the watershed to determine sources of fecal and resistance gene contamination. This analysis found strong correlations of antimicrobial resistance genes with sewer density and septic system age. This indicates that non-point sources of fecal contamination from aging wastewater infrastructure can be critical sources of environmental antimicrobial resistance genes.

6. Detection of beta-lactam resistant Escherichia coli in retail poultry from Egypt. Beta-lactam antibiotics are widely used for treatment of bacterial infections in humans and animals. In Egypt, this class of antibiotics are used for growth promotion and preventative measures in the poultry industry. Food contaminated with beta-lactam resistant bacteria may pose a public health hazard due to the potential of transmission to humans through the food chain. ARS researchers in Athens, Georgia, collaborated with Mansoura University, Egypt, to describe prevalence, antibiotic resistance, phylogroups, and beta-lactamase gene content of Escherichia coli isolates from whole chicken carcasses marketed in Mansoura, Egypt. E. coli was detected in 98% of the chicken carcasses examined, which appeared among the highest contamination levels by E. coli worldwide. Isolates were resistant to multiple antibiotics including three beta-lactam antibiotics. Resistant E. coli were characterized as both pathogenic and commensal. Results from this study are important to both the food safety community and consumers as contaminated retail poultry could be an important source in dissemination of antibiotic resistant strains in Egypt.

7. Discovery of a Listeria monocytogenes heat resistance gene in Listeria innocua. Listeria innocua is considered to be a non-pathogenic species of Listeria. However, similarity of the L. innocua genome with the highly pathogenic Listeria monocytogenes and presence of these bacteria in the same environment may result in gene transfer between the two species. ARS researchers in Athens, Georgia, collaborated with Mansoura University, Egypt, to find virulence genes and genetic components that enable L. innocua recovered from retail milk and dairy products to survive, spread, and cause listeriosis. One antimicrobial resistance gene and thirteen virulence genes were found in the L. innocua but no Listeria specific pathogenic genes. L. innocua isolates also had a gene for heat resistance found on a plasmid. Although this plasmid has been linked to L. monocytogenes that caused a serious outbreak, this is the first report of L. innocua having the heat resistant gene on this plasmid type. The presence of common virulence and antimicrobial resistance genes as well as the heat resistance gene increases the possibility of evolution of virulent strains of L. innocua. This information is useful for the food industry as these strains could challenge processing and preservation protocols and pose health risks from dairy products from Egypt.

8. Emergence of community-associated methicillin-resistant Staphylococcus aureus. Staphylococcus aureus is a Gram-positive bacterium that can be commonly found on the skin or in the nasal passages of most humans and animals. It can cause diseases in humans ranging from minor skin infections to more serious infections such as pneumonia. Community-associated methicillin-resistant S. aureus lineages have appeared in parallel around the world during the past 30 years. ARS researchers in Athens, Georgia, collaborated with researchers from Australia, Papua New Guinea, Germany, Norway, Ireland, Denmark, and Pakistan to find patterns of emergence and spread of community-associated MRSA. Phylodynamic modeling of outbreaks in human host populations and examination of well-recognized epidemic S. aureus lineages revealed periods of abrupt surges in transmission, which typically followed the acquisition of antimicrobial resistance and introduction into a new, often disadvantaged, local host population. Data from the study suggests that antimicrobial resistance, combined with changes in host epidemiology, may have eased the emergence of multiple community-associated S. aureus lineages in the late 20th century. This study supports international collaborative research to monitor emergence and spread of community-associated bacterial pathogens.

Review Publications
Damashek, J., Westrich, J.R., Bateman, J.M., Teachey, M.E., Jackson, C.R., Frye, J.G., Lipp, E.K., Capps, K.A., Ottesen, E.A. 2023. Non-point human fecal contamination from aging wastewater infrastructure is a primary driver of antibiotic resistance in surface waters. Water Research. https://doi.org/10.1016/j.watres.2022.118853.
Cho, S., Jackson, C.R., Frye, J.G. 2023. Freshwater environment as a reservoir of extended-spectrum ß-lactamase-producing enterobacteriaceae. Journal of Applied Microbiology. https://doi.org/10.1093/jambio/lxad034 .
Line, J.E., Seal, B., Garrish, J.K. 2022. Selected antimicrobial peptides inhibit in vitro growth of Campylobacter spp. Peptides. 2(4):688-700. https://doi.org/10.3390/applmicrobiol2040053.
Berrang, M.E., Cox Jr, N.A., Thompson, T.M., Hinton Jr, A., Yeh, H. 2022. Enrichment and direct plating for detection of campylobacter in chicken liver rinse and exudate. Journal of Food Protection. https://doi.org/10.4315/jfp-22-131.
Mcmillan, E.A., Frye, J.G., Weinroth, M.D. 2022. Proliferation of Salmonella Infantis in United States poultry production driven by clonal expansion of a single lineage carrying the pESI plasmid. Microorganisms. https://doi.org/10.3390/microorganisms10071478.
Mcmillan, E.A., Berrang, M.E., Read, Q.D., Ramasetti, S., Richards, A.K., Sharlat, N.W., Frye, J.G. 2022. Buffered peptone water formulation does not influence growth of pESI-positive Salmonella serovar Infantis. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2022.100033.
Ramadan, H., Al-Ashmawy, M., Soliman, A.M., Elbediwi, M., Sabeq, I., Yousef, M., Algammal, A., Hiott, L.M., Berrang, M.E., Frye, J.G., Jackson, C.R. 2023. Whole-genome sequence analysis of Listeria innocua recovered from retail milk and dairy products in Egypt. Frontiers in Genetics. https://doi.org/10.3389/fmicb.2023.1160244.
Yeh, H., Frye, J.G., Jackson, C.R., Read, Q.D., Line, J.E., Hinton Jr, A. 2023. Use of automated capillary immunoassay for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins. Journal of Microbiological Methods. https://doi.org/10.1016/j.mimet.2023.106757.
Abo-Almagd, E., Sabalab, R., Abd-Elghany, S., Ramadan, H., Jackson, C.R., Sallam, K. 2023. ß-lactamase producing Escherichia coli encoding blaCTX-M and blaCMY genes in chicken carcasses from Egypt. Foods. https://doi.org/10.3390/foods12030598.
Steinig, E., Aglua, I., Duchene, S., Meehan, M., Yoannes, M., Firth, C., Jaworski, J., Drekore, J., Urakoko, B., Poka, H., Wurr, C., Ebos, E., Nangen, D., Mueller, E., Mulvey, P., Jackson, C.R., Blomfeldt, A., Vangstein Aamot, H., Laman, M., Manning, L., Wirth, T., Earls, M., Coleman, D., Greenhill, A., Ford, R., Stegger, M., Syed, M., Jamil, B., Monecke, S., Ehricht, R., Smith, S., Pomat, W., Horwood, P., Tong, S., Mcbryde, E. 2022. Phylodemographic signatures in the emergence of community-associated MRSA. The Lancet Microbe. https://www.pnas.org/doi/epdf/10.1073/pnas.2204993119.