Turkeys.

2022-23 Rapid Ag: Autochthonous Bacteriophages for Preharvest Safety of Turkeys: Integrating Salmonella’s Phage Immune Determinants (SaPhIDs) to Phage Therapy Design

March 1, 2021

Project Leader

Anup Kollanoor Johny, Department of Animal Science

Team Members and Roles

  • Tim Johnson, Department of Veterinary and Biomedical Sciences
  • Sally Noll, Department of Animal Science
  • Carol Cardona, Department of Veterinary and Biomedical Sciences
  • Luna Akhtar, Department of Animal Science
  • Petri Papinaho, VP, Research & Development, Jennie-O Turkey Store

Non-Technical Summary

Problem: Turkeys can harbor Salmonella in the guts, resulting in the fecal-oral spread of the pathogen to incoming turkeys, ultimately contaminating products. The recent outbreaks through turkey products of Salmonella Reading, a rare serotype causing outbreaks, is surprising and alarming, and indicates the emergence of new types of Salmonella in turkey production. Due to the federal initiatives to curb antibiotic use, alternatives such as bacteriophages (viruses that predate on pathogenic bacteria) are being explored for their anti- Salmonella activity.

Discovery with Industry Partnership: Our recent studies indicate that turkey-specific lytic DNA bacteriophages inhibit drug-resistant Salmonella on turkey skin. Our ongoing Minnesota Turkey Growers Association (MTGA)-funded project testing these phages against emerging Salmonella is an example of industry-academia partnership developing collective solutions.

Need for Rapid Response Funds: This proposal aims at using the samples from the MTGA-funded project for in-depth multi-omics analyses, specifically studying and integrating Salmonella’s phage resistance, to developing robust and next-generation science-powered bacteriophage selection toolkits against Salmonella.

Education: We will educate the producers, processors, and consumers on emerging bacteriophage therapies to fight the devastating effects of Salmonella on Minnesota’s turkey industry.

Objectives and Goals

The overall goal of this comprehensive, integrated, multi-college, multi-departmental project is to assimilate Salmonella’s Phage Immune Determinants (SaPhIDs) in developing integrator-friendly, flock/farm-derived (autochthonous) bacteriophage therapy against emerging Salmonella serovars in turkeys (Salmonella Reading) powered by a multi-omics approach. This request is made explicitly for the genomic-, phenomic-, and microbiome- analyses of samples from the ongoing MTGA project determining the efficacy of farm-derived custom-made bacteriophage cocktail therapies against Salmonella to guide better understanding of the mode of action of these products. This research must be immediately undertaken to design a multi-omics science-based selection strategy for phage products intended for turkey production, otherwise potentially having long-term adverse impacts due to the persistence of phage-resistant Salmonella on turkey farms.

Hypothesis: Application of lytic DNA bacteriophage cocktails will reduce S. Reading in the turkey cecum by downregulating SaPhIDs and virulence. There will be significant changes in the cecal microbiome correlating with S. Reading challenge and bacteriophage therapy, guiding and enhancing mitigation strategies. The end-user education will improve knowledge on sustainable bacteriophage-based therapies against Salmonella for turkey growers and consumers and comfort level using such products.

Objectives:

  1. To explore the antibacterial effect of lytic DNA bacteriophages against S. Reading, mechanistically, using genomicand phenomic- analysis of SaPhIDs and virulence, and gut health-focused microbiome analysis
  2. To disseminate project results (applied information from the ongoing MTGA project and mechanistic information from this proposed project) and educate turkey growers and the public.

Discovery: Integrating novel Salmonella phage immune genes, rapid phenomic tests, and bacterial gut-health markers to the phage designing toolkit for better bacteriophage therapies against Salmonella.

Justification

The problem of Salmonella: Contamination of turkey products with Salmonella has historically resulted in product recalls causing enormous losses to the turkey industry. Turkey burgers, ground turkey, and raw turkey products have been implicated in multistate outbreaks caused by S. Hadar (2011), S. Heidelberg (2011), and S. Reading (2018), respectively 1,2,3. Salmonella colonizes all segments of the turkey gastrointestinal tract, most notably the ceca. Excretion of the pathogen through feces results in infection of incoming flocks and farm contamination. Besides, a faulty processing step could result in cross-contamination of carcasses.5,15 It is well established that Salmonella mitigation is a moving target, highly dependent on on-farm Salmonella ecology. These situations warrant Minnesota’s turkey industry to accelerate efforts to find rapid, science-based solutions against Salmonella.

Bacteriophages as preharvest biocontrol strategy against Salmonella: The uniqueness of bacteriophages (viruses that predate on bacteria) is attributed to their highly specific bacterial-kill kinetics, high bacterial host specificity, lack of animal toxicity, self-replicating and self-limiting abilities, ubiquitous distribution in nature, and negligible effects on food quality. Phages undergo two primary life cycles – lytic and lysogenic. In the lytic cycle, which we plan to focus on in this research, phages adsorb to the bacterial cell receptor (lipopolysaccharide (LPS), flagella). After adsorption, the viral nucleic acid is injected into the bacteria. The bacterial machinery begins replicating viral genetic material. This follows self-assembly of the viral proteins and formation of capsid head. After tail assembly, they will lyse the bacterial cell wall, releasing the progeny to the environment 6,16. This is how phages kill the bacteria.

Salmonella’s resistance to phages – a hurdle in designing therapies: A significant challenge in finding a bacteriophage-based solution is the hurdle of resistance build-up in Salmonella to their specific phages. Salmonella’s resistance (immunity) to phages is different from their resistance to antibiotics. Bacteria have evolved diverse defense strategies to evade phage infection or lysis. These strategies include immunity by a surface modification to resist attachment of phages on to the bacterial surface, blocking phage DNA injection into bacteria (superinfection exclusion), cleavage of phage genetic material inside bacterial cytoplasm, and bacterial suicide reaction upon phage infection (abortive infection)16. Understanding resistance development in Salmonella to phages from an applied industry hurdle perspective is needed for developing sustainable phage-based solutions for various industry applications. Recently, few companies have launched phage products for use in poultry production. We are collaborating with a global phage manufacturer (Proteon Pharmaceuticals; letter attached) and testing their product in the ongoing study as an industry control. This collaboration has been consulted with the Minnesota turkey integrator, Jennie- O, signifying the industry’s interest.

Our approach: We are utilizing a two-pronged approach: (1) determining the biocontrol efficacy of custom phage cocktails from turkey farms and (2) determining the nature of phage-pathogen interactions at the pathogen and gut levels. Together, these data will add power to select the right phage cocktails as determined by next-generation Omics-based science for various turkey industry applications. Based on our recent finding that turkey-specific lytic DNA bacteriophages (Figure on the right - top) inhibit a drug-resistant Salmonella on turkey skin at chilling (Figure on the right - bottom), MTGA funded our first approach. It will address several applied questions, including dose, timing, appropriate delivery method, all validated by turkey challenge studies. This Rapid Response proposal targets the use of invaluable samples from the MTGA-funded research for Omics investigations [(1) genomics, (2) comparative phenomics, and (3) microbiomics] that will further inform the intelligent development of sustainable phage-based therapies in turkeys.

  1. Genomic analysis of SaPhIDs and virulence expression in S. Reading: A global transcriptome analysis using RNA-seq will determine the overall changes in mRNA expression of S. Reading exposed to bacteriophage therapy in 12-week old turkeys17. RNA-seq analysis has been a powerful tool for developing new drugs by investigating overall gene structure patterns and function patterns. In our study, this is highly relevant as reports studying the role of SaPhIDs in bacteriophage therapies in poultry, especially profiling the global changes/regulation and metabolic and virulence pathways in Salmonella, are lacking. Based on our finding with S. Heidelberg, we hypothesize that bacteriophages down-regulate SaPhIDs and virulence, including the core genes present in Salmonella. Rapid Outcome: A set of SaPhID genes added to the phage selection toolkit.
  2. Comparative phenome analysis of bacteriophage-stressed S. Reading: This step examines the potential of post-challenge S. Reading stressed due to bacteriophage treatments to result in altered virulence and environmental survival capabilities. We will test postchallenge S. Reading with and without bacteriophage exposure for their phenotypic expression of adhesion, invasion, motility, time-kill kinetics, antibiotic susceptibility21, and phage adsorption to know if they have an altered phenotype 9,10,12. Rapid Outcome: Set of benchtop tests to study and customize potential phage candidates by knowing their interactions with the host Salmonella.21
  3. Microbiome analysis to identify bacterial signatures of phage- S. Reading interactions in the turkey gut: In the past Rapid Response projects, we conducted microbiome analysis of multiple alternatives to antibiotic (A2A) interventions (probiotics, prebiotics, vaccination) and have characterized microbial signatures of the healthy and compromised gut in response to MDR S. Heidelberg challenge. We have found unique bacterial signatures for Salmonella colonization and A2A interventions (several talks have been given; documentation in progress). In this study, we plan to obtain information on the effects of pathogen challenge and bacteriophage intervention on bacterial community structure and the communities most susceptible to infection and phage supplementation. Rapid Outcome: Development of a comprehensive microbiome-signature table (bacterial markers of gut health) in relation to different A2A interventions to design intelligent and sustainable turkey gut-health friendly bacteriophage cocktails 4,11.

Short and Long-Range Cost-Benefit Potential: Our approach will identify bacteriophage cocktails against Salmonella with evidence on their action mechanisms to produce a long-term sustainability footprint for the turkey industry. We will also obtain information on how to address Salmonella resistance to phages from an Omics perspective. Bacteriophages could enrich a unique group of bacteria and will add to the comprehensive table of target bacterial populations for devising intelligent Salmonella control methods. Furthermore, changes to the SaPhID and virulence genes in response to phages are expected, a finding with significant value to developing future farm-specific phage selection methods. Our group has won federal grants utilizing the data generated from such feeder projects.

References

  1. CDC. http://www.cdc.gov/salmonella/heidelberg/index.html
  2. CDC.http://www.cdc.gov/salmonella/hadar0411/040411/index.html?s_cid=ccu04111 1_016
  3. CDC. https://www.cdc.gov/salmonella/reading-07-18/index.html
  4. Danzeisen et al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3883494/
  5. EFSA. https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2012.2616
  6. Gill and Hyman. https://pubmed.ncbi.nlm.nih.gov/20214604/
  7. Janssens et al. https://aem.asm.org/content/74/21/6639
  8. Kaeothip et al. https://pubs.rsc.org/en/content/articlehtml/2011/ra/c1ra00145k
  9. Johny et al. DOI: 10.1128/AEM.07643-11
  10. Johny et al. https://www.frontiersin.org/articles/10.3389/fmicb.2017.01828/full
  11. Nair et al. https://poultrysci.morwebcms.com/files/galleries/2018-PSA-Abstracts.pdf (529P)
  12. Nair, & Johny. https://doi.org/10.3389/fmicb.2018.01475
  13. Roer et al. https://msystems.asm.org/content/1/3/e00009-16
  14. Rojas et al. https://pubmed.ncbi.nlm.nih.gov/11682190/
  15. Rostagno et al. https://academic.oup.com/ps/article/85/10/1838/1534332
  16. Seed et al. https://pubmed.ncbi.nlm.nih.gov/26066799/
  17. Shah. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911198/
  18. Splichalova et al. https://www.mdpi.com/2072-6651/11/9/534/htm
  19. Wahl et al. https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.14167
  20. Akhtar et al. https://doi.org/10.1016/j.foodcont.2013.09.064
  21. Chan et al. https://www.nature.com/articles/srep26717