CHARACTERIZATION AND GENE-EDITING OF BACTERIOPHAGES AS BIOCONTROL AGENTS OF FOODBORNE PATHOGENS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016749
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Food Science & Nutrition

Non Technical Summary
Salmonellahas been associated with diverse food products including poultry, meat, eggs, fresh produce and dried foods. Many of theSalmonellastrains isolated from foods are resistant to multiple antibiotics and this could be correlated with the heavy use of these important drugs in agriculture. Also, fresh produce foods that have been associated withSalmonellaoutbreaks, cannot be thermally processed to kill the bacteria prior to human consumption. In both of these food sectors, there is a demand for a safe and non-destructive method to controlSalmonellain foods. One option that is being explored is to apply to the food or animal, a targeted antimicrobial that specifically kills the pathogen of interest without affecting the other microorganisms that might be present. These antimicrobials are called bacteriophages, a class of viruses that are only capable of killing bacteria and do not harm humans, animals or plants. Several bacteriophage products have already received approval by the FDA to kill specific pathogens such asSalmonellaandListeriain food products and processes. However, the effectiveness of these approaches has not been fully explored. One concern is that bacteria can acquire resistance to bacteriophage that would limit their effectiveness in controlling food borne pathogens. Fortunately, bacteriophage can overcome bacterial defense systems with their own gene products. Thus, bacteria and bacteriophage are locked in an evolutionary arms race that we must further understand if we are to heavily utilize bacteriophage in food and agriculture.To address this problem, this project will identify the mechanisms thatSalmonellautilize to protect themselves from bacteriophage and the counter systems that bacteriophage use to overcome them. We will combine this gleaned knowledge with genetic engineering tools to produce bacteriophage that kill a broader range ofSalmonellastrains, and thus will be safer to use within the food and agricultural industries. These studies will help to future proof the application of bacteriophages in the food industry and protect us from using them irresponsibly and leading to problems of bacterial resistance. At the end of the planned series of experiments, the results will be presented at national meetings and manuscripts will be prepared for publication in the scientific literature. Data will be available for risk assessors and regulatory agencies.

Animal Health Component

20%

Research Effort Categories

Basic

50%

Applied

20%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

food safety

microbiology

salmonella

bacteriophage

crispr

bacteria

gene editing

genetics

bacteriophage resistance

alternatives to antibiotics

Goals / Objectives
The long-term goal of this project is to characterize and gene-edit bacteriophages so that they can be effectively used as biocontrol agents of foodborne pathogens. We plan to conduct a series of experiments with Salmonella and its bacteriophages to understand the "arms race" that exists between them. Specifically, we will investigate bacterial immunity to phage and the corresponding counter-measures employed by phage to overcome bacterial immunity systems. We will exploit this knowledge to genetically engineer enhanced bacteriophages that could be more effective in eliminating Salmonella from foods and food production environments.The specific objectives are:1) Identify and characterize Salmonella genes that confer immunity to a broad host-range lytic bacteriophage.2) Identify and characterize bacteriophages that possess genes that overcome bacterial phage immunity systems.3) Use genetic engineering approaches to construct broad host range bacteriophages that are resistant to Salmonella's phage defense systems and assess if they are more effective in eliminating the pathogen from foods.

Project Methods
This project will be conducted by performing a series of microbiological, biochemical and molecular biology experiments using pure cultures of Salmonella strains isolated from foods and bacteriophage isolated from sewage samples or purchased from culture collections. The Food Safety Microbiology Laboratory at Andrew Boss Laboratory of Meat Science is a biosafety level 2 facility fully equipped to conduct bacteriological research. The experimental approach is described for each of the objectives below.Objective 1We will screen bacteriophage Felix O1 against a collection ofSalmonellastrains isolated from raw meat and poultry samples on xylose lysine deoxycholate plates. Serial dilutions of Felix-O1 phage lysates will be spotted onto top agar lawns seeded with eachSalmonellastrain. Strains that show evidence of resistance to Felix-O1 at lower multiplicities of infection will be further characterized as potentially encoding resistance mechanisms against bacteriophage. Three approaches will be taken to identify these mechanisms of resistance, A) prophage identification, B) fosmid library screening and C) transposon mutagenesis.For A), mitomycin-C will be used to induceSalmonellaendogenous prophage to enter the lytic cycle and then tested for their capacity to lysogenize Felix-O1 sensitive strains and confer resistance to Felix-O1. The prophages will be characterized using electron microscopy (UMN Characterization Facility) and DNA sequencing (UMN Genomics Center) to identify and classify them.For B)Salmonellagenomic DNA will be processed into fosmid libraries using commercial kits and transformed intoEscherichiacoliprior tolambda-phage transduction into Felix-O1 sensitiveSalmonellastrains. Cosmids that confer resistance to Felix-O1 will be DNA sequenced to identify the genes responsible for resistance to Felix-O1.For C), random transposon mutagenesis will be performed on Felix-O1 resistantSalmonellaand analyzed for conditional lethality using TnSeq using the DNA sequencing facilities available at the UMN Genomics Center.Objective 2Two approaches will be used to identify phage genes that counter Salmonella phage immunity genes; A) phage co-infection and direct selection forinvivotransfer of resistance genes and B) chemical mutagenesis of phage.For A), Felix-O1 resistantSalmonellastrains will be co-infected with Felix-O1 and additional bacteriophage that will be isolated from sewage samples that lyse the Felix-O1 resistant strains. During co-infection experiments, genetic rearrangements may occurinvivoto generate chimeric phage that are comprised of genes from both Felix-O1 and another phage. Broad host range chimeric phages that are able to propagate onSalmonellathat are ordinarily resistant to Felix-O1, will be sequenced to identify the genetic rearrangements responsible for the enhanced resistance toSalmonellaphage immunity systems.For B), we will apply hydroxylamine chemical mutagenesis of bacteriophage DNA, to identify genes that are conditionally required for propagation on Felix-O1 resistantSalmonellastrains. Hydroxylamine can mutate phage DNA whilst packaged within phage particles and can generate stop codons within genes that lead to gene inactivation. Any essential phage gene that is inactivated by the chemical mutagen will prevent the phage from being able to replicate. Using DNA sequencing and comparing phage replication on Felix-O1 resistantSalmonellastrains against Felix-O1 sensitive Salmonella strains, we will identify genes that protect phage fromSalmonellaphage immunity systems.Objective 3In this objective we will use CRISPR-Cas9 methods to alter the host range of members of thefelixO1phage genus. Specifically, we will target the tail fibers within these phage to determine if host ranges can be altered by swapping phage tail genes and adsorption to host bacterial cells. We will also determine if phage genes identified in objective 2, which confer resistance againstSalmonellaphage immunity systems, can be introduced into Felix-O1 and other members of its genus. This will determine if the host range of bacteriophages can be expanded to overcome bacterial defenses against phage.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:A seminar entitled "The study of foodborne bacterial pathogen stress responses using transposon sequencing (TnSeq)," was given by Steven Bowden on November 6th 2018 at the Dept. of Food Science, Purdue University. A seminar was given to at the University of Minnesota Dept. of Food Science showcase on April 22nd, 2019. I also participated in the Sept. 2019 meeting for the S1077: Enhancing Microbial Food Safety by Risk Analysis meeting at the University of Minnesota. These were opportunities to show research in the lab to other experts interested in food safety. Changes/Problems:This year our lab space had to be relocated due to essential renovation work that had to be performed on the Andrew Boss Laboratory of Meat Science building. This has been disruptive for our efforts this round and remains a constraint on our current space and facilities. We will relocate to our original lab space in May 2020 which will improve our access to microbiological equipment and provide us more space to work on this project. What opportunities for training and professional development has the project provided?At this time, this project is forming the basis of 2 MSc graduate students research projects. One of them is a Food Science Major whilst the other will be pursuing a degree in Microbial Engineering. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?For objective 1, we will perform adsorption assays on Salmonella strains that do not form PFUs with Felix-O1 to determine if they have poor adsorption. If adsorption is comparable to sensitive strains, then we will consider investigating these strains further by generating a genomic fragment library of clones and selecting for the genes that can protect Salmonella from Felix-O1. To characterize the abortive infection system further, we will assay for whether adsorption or burst size is reduced when Salmonella expresses the system. This will determine if the mechanism of resistance is post-adsorption which would agree with previous literature on abortive infection systems. For objective 2, we will determine whether Felix-O1 can evolve to escape the abortive infection system by propagating the phage on Salmonella that possess the system. If a spontaneous mutant phage can be identified that efficiently infects strains that express the abortive infection system, we will DNA sequence the phage DNA to isolate the mutations. In parallel, we will generate a Salmonella LPS mutant that we can infect with VpaE1, which is one of the Felix-O1-like phages that is immune to the abortive infection system. This will allow us to determine if this phage is also immune to the abortive infection system when infecting Salmonella in case the abortive infection is non-functional in E. coli. If found to still be resistant, we will clone the genes present in VpaE1 and absent in Felix-O1 to determine whether their expression in Salmonella enhances sensitivity to Felix-O1. We will also start to test co-infection experiments with Felix-O1 and our newly isolated phages to see if chimeric phages can be generated through recombination between phage genomes leading to new phages with altered or expanded host ranges. For objective 3, we will continue to test whether the myTXTL system can be applied to the Felix-O1 family of phages. If successful, we will test whether in vitro we can perform recombination and transposition reactions to modify the DNA. In parallel, we will test in vivo systems for introducing DNA during Felix-O1 infection of Salmonella. Techniques that will be tested are lambda red recombination and transposition. As proof of principal we will attempt to introduce easily detectable markers such as GFP or luciferase. We will determine whether the antitoxin gene from the abortive infection system can serve as a selection marker for these techniques to facilitate genetic engineering of Felix-O1.

Impacts
What was accomplished under these goals? During this reporting period the following progress has been made in regards to the stated objectives of this project. 1) Identify and characterize Salmonella genes that confer immunity to a broad host-range lytic bacteriophage. We have screened strains of Salmonella from serotypes Typhimurium, Enteriditis, Reading, Infantis and 1,4,[5],12:i:- for sensitivity to the broad Salmonella host range bacteriophage, Felix-O1. All Salmonella strains so far tested display sensitivity to this phage in spot tests although the ability to form visible plaque forming units (pfu's) can vary. Some strains only display visible lysis when the concentration of phage is high and never generate visible pfu's. These strains may have mechanisms of resistance to Felix-O1 that include poor adsorption and/or post adsorption immunity systems. For some of these strains, pfu's can only be observed in response to increased NaCl or glycerol concentration. A possible explanation for this could be that faster bacterial growth in the assay leads to more rapid phage generation and thus visible pfu's. Alternatively, NaCl and glycerol may influence phage adsorption to these strains of Salmonella and this may indicate that the efficiency of pathogen control with this bacteriophage is dependent on the composition of the food matrix itself. We will seek to address whether adsorption is influenced by glycerol or NaCl against these Salmonella strains. For four S. Enteriditis strains, we identified a plasmid that encodes a type-3 phage abortive infection system. These systems are comprised of an RNA antitoxin that inhibits a growth inhibitory protein toxin and they are known to antagonize phage replication. Although Felix-O1 can lyse these four strains of S. Enteriditis at a high titer, it is unable to form visible pfu's on them. We considered that the abortive infection system could confer resistance to Felix-O1 and therefore subcloned the toxin-antitoxin genes from the plasmid into a plasmid vector and tested them for protection against Felix-O1. We observed that S. Typhimurium LT2, a strain ordinarily sensitive to Felix-O1, had increased resistance to this phage when it possessed a plasmid with the abortive infection system. Specifically, we observed around a 2-fold reduced efficiency of plating and that the pfu's were almost invisible unless glycerol was added to the medium. This could suggest that the phage replication is being inhibited post adsorption including a reduced burst size. To our knowledge this is the first known example of a system in Salmonella that confers protection against Felix-O1 through an abortive infection mechanism. It remains possible that other resistance mechanisms are present in the Salmonella strains in our lab. As a first step we will perform adsorption assays to determine if their resistance is due to poor adsorption. Where adsorption is not the mechanism of resistance, we will investigate the strains further for post-adsorption resistance mechanisms. 2) Identify and characterize bacteriophages that possess genes that overcome bacterial phage immunity systems. As discussed above, the Salmonella specific bacteriophage, Felix-O1, is sensitive to an abortive infection system present in some strains of Salmonella. We also tested Escherichia coli specific members of the Felix-O1-like bacteriophage family for sensitivity to the abortive infection system we identified. Interestingly, these bacteriophages are not sensitive to the abortive infection system. We are investigating what the basis for this could be and whether gene differences between these bacteriophages could account for differential sensitivity to the abortive infection system. We have also been isolating bacteriophages from wastewater samples that infect strains of Salmonella in our lab. Filter sterilized wastewater is added to specific Salmonella strains to enrich for the bacteriophages in that sample that can replicate on the bacterial strains. We have isolated several bacteriophages this way and have been testing their host ranges on our laboratory Salmonella strains. At this time, we have not isolated phages with as broad a host-range as Felix-O1 but several phages can infect strains that do not develop pfu's with Felix-O1. We will investigate these phages further to determine if they are lytic or lysogenic and sequence their DNA to further characterize them. 3) Use genetic engineering approaches to construct broad host range bacteriophages that are resistant to Salmonella's phage defense systems and assess if they are more effective in eliminating the pathogen from foods. In collaboration with Prof. Vincent Noireaux (University of Minnesota), we are investigating whether bacteriophages such as Felix-O1 can be produced in vitro. Such a method will provide a platform to perform genetics on bacteriophage using in vitro molecular biology methods. We have developed procedures to isolate Felix-O1 genomic DNA in sufficient quantities to use with the myTXTL in vitro expression system. We were able to produce T7 bacteriophage using this method but have not yet been able to produce Felix-O1 pfu's using this system. We are currently still working on this method. If successful, we will move towards introducing DNA into Felix-O1 such as the antitoxin gene described above to generate a recombinant phage that is able to more efficiently infect Salmonella hosts that possess this phage immunity system.

Publications