SEED GRANT: PHAGE ENDOLYSINS AS DISINFECTANT AGAINST LISTERIA MONOCYTOGENES ON SPINACH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1029912
• Grant No.: 2023-67018-39268
• Project No.: TU-1-Sharma 2022
• Proposal No.: 2022-09027
• Program Code: A1332
• Project Start Date: Aug 1, 2023
• Project End Date: Jul 31, 2025
• Grant Year: 2023

Project Director
Sharma, A.

Recipient Organization
TUSKEGEE UNIVERSITY
(N/A)
TUSKEGEE,AL 36088

Performing Department
(N/A)

Non Technical Summary
Listeria monocytogenes (Lm) is a human pathogen and world-wide, the leading cause of bacterial linked foodborne mortality. Outbreaks of listeriosis affect primarily at- risk people (pregnant women, elderly, immunocompromised individuals) and can be traced to consumption of smoked meats, seafood, and dairy products as well as preharvest fresh produce (cabbage, corn, lettuce, celery, radish, and spinach) and the presence of Lm in the farm environment. Antibiotics have been used to inhibit the growth of pathogenic bacteria for many decades, reducing their threat to human health. However, due to the increasing number of antibiotic resistant and multi-drug resistant strains of bacteria it has become necessary to explore new alternatives for eradicating pathogens. Bacteriophage (phage) endolysins are enzyme antimicrobials that digest the major building blocks of the bacterial cell wall. In Gram-positive bacteria including Lm, the cell wall is surface exposed, so the purified endolysin when exposed externally, can cause death of the pathogen by degrading the cell wall. Endolysins are highly species-specific and refractory to resistant strain development. Despite numerous efforts to find them, not a single endolysin-resistant bacterial strain has been identified so far. We believe that endolysins can prevent or eradicate Lm on leafy vegetables. This proposal will examine the known Lm phage endolysins for their efficacy against Lm strains found on spinach. However, endolysin purification is not cost-effective for treating large quantities of leafy vegetables with a purified Lm phage endolysin, and this treatment alone would not control other food-borne bacterial pathogens such as Salmonella or E. coli. To address this concern, current treatments to eradicate food borne pathogens include chemical sanitizers. However, food borne pathogens have been reported to develop resistance to chemical sanitizers, indicating that Lm might also eventually develop resistance to these chemical agents. Thus, it is the goal of this project to identify individual or paired Lm endolysins with the highest efficacy against spinach born Lm and to then test for increased killing efficacy by pairing the endolysin treatment with existing food industry chemical treatments. This should identify a more sustainable treatment of food borne pathogens that does not incur significantly increased treatment costs while reducing the risk of creating treatment-resistant strains of Lm.

Animal Health Component

0%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1430	1040	50%
511	4010	1100	50%

Knowledge Area
511 - New and Improved Non-Food Products and Processes; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

antimicrobial

food safety

listeria monocytogenes

phage endolysins

and spinach

Goals / Objectives
The goal of this project is toidentify cold-active Lm endolysins and to test for endolysin-endolysin and endolysin-sanitizer synergies to find a more efficacious Lm treatment on spinach with minimal cost increases.Specific Aims:Aim 1. Compare cold-active Lm phage endolysins (e.g. LysZ5, Ply511, PlyP100, PlyP40, PlyPSA) against food safety strains of Lm at room temperature (RT) and food processing temps.Aim 2. Compare the optimal phage endolysins against food safety strains of Lm on spinach at RT and at post-harvest processing temperatures.Aim 3. Test for synergy against Lm between the most active endolysins and food sanitizers at RT and on spinach at post-harvest processing temps.

Project Methods
AIM 1. Compare cold-active Lm phage endolysins (e.g. LysZ5, Ply511, PlyP100, PlyP40, PlyPSA) against food safety strains of Lm at RT and food processing temps.Aim 1.1. Use standard peptidoglycan hydrolase (PGH) assays to assess the relative activity of each representative endolysin on multiple strains of Lm. Plasmids expressing the 6xHis tagged endolysin enzymes will be obtained from either Mathias Schmelcherat the ETH,or will be synthesized commerciallywith E. coli codon bias, 6 x His tag, in pET21a inducible vector [in E. coli BL21(DE3)]. Purity via nickel chromatography will be verified via SDS PAGE and tested against the major US food safety Lm strains.Multiple assays will be used to verify the relative activity of the candidate enzymes at room temp. These include the plate lysis, turbidity reduction, zymogram,and the MIC.Aim. 1.2. Test all Lm phage endolysins for Lm strain specificity and activity at food processing temperatures (5 and 10 ºC) using the same assays as in Aim 1.1. Food safety Lm strains will be grown to mid log phase, and chilled on ice to the appropriate temperature for the assay. The most active endolysins with the broadest range of activity will be used in Aim 2.Aim 1.3Test Lm phage endolysins for pairwise synergy.Synergy testing will be as for LysK and lysostaphin against Staphylococcus aureus in a modified MIC assay (Becker et al., 2008).AIM 2. Compare the optimal Lm phage endolysins against food safety strains of Lm on spinach at RT and at post-harvest processing temperatures.Fresh spinach from a local grocery, with the same expiration date, will be purchased and stored at 5 ºC for < 24 h prior to use. 5 g of healthy-looking greens will be weighed and dispensed into sterile stomacher bagsbefore use. This will compare the efficiency of endolysin against Lm on spinach at different temperatures.Aim 2.1. Test for background levels of pre-existing Lm. Perform four independent replicates. Combine 45 ml of DIFCO buffered peptone waterwith 5 g of fresh spinach in a 0.532 L Whirl Pak® bag Pummel by hand (~10 min) until the greens are reduced to small pieces, exposing the internal leaf structure. Perform serial dilution of thesolution in triplicate in 0.1% peptone water (PW) diluent, plated on Oxford Listeria-selective agar with Oxford Listeria-selective supplement, count CFUs after 24 h at 37 ºC.Aim 2.2. Prepare spinach inoculated with Lm. Inoculate 5 gm portions of greens in sterile stomacher bagswith 0.5 mL of 102 CFU/mL Lm inoculum in PBS. Shake the bags ~30 times to disperse the inoculum. Nine bags are prepared for each sampling time and the experiment is performed in triplicate. The Lm inoculated samples are then placed in an incubator and maintained at constant temperature (5, 10, or 25 ºC) for the experimental period of time. Control samples will be treated with PBS buffer alone, (no Lm inoculum).Aim 2.3. Endolysin treatment of Lm inoculated spinach. Each experimental group contains nine bags of fresh spinach (5 g per sample) inoculated with 0.5 mL of the 102 CFU/mL (Lm). There are 5 groups (G1-G5) consisting of: G1), G2), G3) 9 bags each stored at three different temperatures (5, 10, or 25 ºC) washed with 45 ml pre-cooled endolysin solution [20 mL purified endolysin, 80 mL PBS), G4) 9 bags washed with PBS buffer alone (negative control) and G5) 9 bags washed with 200 mg/L of chlorinated water (positive control) at pH 7.0 for 10 min at room temperature. During the treatment, the washing solution was occasionally stirred. And then each 5 x 9 bags (45 x 5g samples) are placed inbag and kept at 5, 10, or 25 ºC for 2 h. Enumeration of Lm microorganisms on the greens will be as in Aim 2.1.Aim 3. Test for synergy against Lm between the most active endolysins and food sanitizers at RT and on spinach at post-harvest processing temps.Aim 3.1 Using cold-active Lm endolysins, determine their optimum pH via turbidity reduction assay and pair with sanitizer(s) with a similar active pH range. Using the turbidity reduction assay protocol of Swift et al., 2018 with several buffers to cover a broad pH range, identify the activity level of each endolysin at a range of pH values. Match the sanitizerto endolysins that share a pH with at least 50% full antimicrobial activity.Aim 3.2 Determine the concentrations of the endolysin and sanitizer (for each endolysin/Detergent pair identified in Aim 3.1) that does not inactivate the endolysin. Using the pairs of endolysin and detergent identified in Aim 3.1, we will start with the highest concentration of endolysin that we can produce via nickel column chromatography (Aim 1) in an appropriate pH buffered solution (compatible to optimize both the endolysin and sanitizer). Keeping the reaction volume less than 100 µL we will start with a reaction condition that reflects the optimal exposure time, temperature (hopefully 4ºC) and detergent concentration per manufacturers recommendation. At the appropriate time of exposure, the 100 µL sample will be loaded onto a Bio-Rad Micro Bio-Spin™ P-6 Gel Columnsthat has been pre-equilibrated with an appropriate buffer, and pre-spun to remove excess buffer. These columns remove compounds <6 kD by size exclusion chromatography. The largest of the sanitizer is the quaternary ammonium salts with a MW of ~550. The effluent from the spin column will harbor the sanitizer treated endolysin (~25-50 kDa) without any sanitizer in an appropriate buffer at the endolysin optimal pH. We will then determine the effluent endolysin concentration (OD280 nanodrop) and use the endolysin assays in Aim 1 to determine if there is a loss of activity after exposure to the sanitizer. The control will be untreated endolysin (mock assay) that was purified over an identical spin column. This strategy will be repeated with rational design to determine at what sanitizer concentration/time of exposure/temperature that the endolysin activity loss is minimized by exposure to the sanitizer.Aim 3.3 In order to test feasibility for industrial applications, determine at what minimal concentration of endolysin and sanitizer we can identify antimicrobial synergy in vitro.Starting with a cohort of 50 ml tubes harboring 20,000 CFU Lm in 0.2 ml of buffer at 4ºC.To this cohort of tubes add diminishing concentrations of endolysin (<10 µL volume) such that the mixture can be readily diluted with 19.8 mL of buffer and 0.1 ml of this diluted reaction plated directly onto agar plates (expected 200 CFU per plate) as a means to simultaneously stop the assay and perform a plating assay. We predict that a 100X dilution will effectively "reduce to the point of negligible activity" both the endolysin and the sanitizer for the time it takes to plate the 0.1 ml sample, any residual endolysin or sanitizer will then be diluted further on the agar plate. All samples and plating will be performed in triplicate. The tube just described will be an endolysin alone control sample. To additional identical tubes, add increasing concentrations of sanitizer while keeping the endolysin concentration constant and incubate at the appropriate exposure times [in keeping with the results of Aim 3.2 (compatible endolysin and sanitizer concentrations, exposure times, temperatures)]. Each tube with a different endolysin concentration will have an endolysin alone control with the same endolysin concentration, and each tube with a different sanitizer concentration will be supported by a sanitizer alone control at the same sanitizer concentration. Plotting of CFUs resulting from this assay alongside the endolysin and sanitizer concentrations utilized, should reveal at which point (if any) the combination of endolysin and sanitizer are more than additive.Aim 3.4 Translate the optimal synergistic conditions determined in Aim 3.3, to spinach as in Aim 2.2, to identify the optimal synergistic conditions for use on spinach.

Progress 08/01/23 to 07/31/24

Outputs
Target Audience:Our target audience includes undergraduate and graduate students from theTuskegee University. During the study period, we mentored three undergraduate students and one Ph.D. student. The undergraduate students received training in essential microbiology and molecular biology skills, including aseptic techniques, bacterial culturing, handling Listeria monocytogenes (Lm), performing plate lysis assays, plasmid purification and transformation, gel electrophoresis, and other foundational laboratory techniques. The Ph.D. student actively worked on this project, successfully expressing three endolysin proteins--Ply511, PlyP40, and PlyPSA--through IPTG induction. She conducted multiple microbicidal assays, such as plate lysis, turbidity tests, and Lm growth inhibition, to evaluate the activity of the crude extracts of these endolysins against Lm. Furthermore, she successfully purified the PlyPSA endolysin using nickel column chromatography, verifying its purity with SDS-PAGE analysis. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three undergraduate research students participated in this project under PDs mentorship andreceived training in essential microbiology and molecular biology skills, including aseptic techniques, bacterial culturing, handling Listeria monocytogenes (Lm), performing plate lysis assays, plasmid purification and transformation, gel electrophoresis, and other foundational laboratory techniques. Using Course Based UndergraduateResearch Experience (CURE) model PD trained 40 studenst in two sections of "Molecular Cell and Genetic Biology" Laboratory. Students learned basicmicrobiology and molecular biology skills and scucessfully purified plasmid vectors carryingPly511, PlyP40, and PlyPSA and transformed E. coli competent cells with these plasmid vectors. One Ph.D. student is actively workingon this project under PDs supervision. She received training in essential laboratory skills and successfully expressedthree endolysin proteins--Ply511, PlyP40, and PlyPSA,. She conducted multiple microbicidal assays, such as plate lysis, turbidity tests, and Lm growth inhibition, to evaluate the activity of the crude extracts of these endolysins against Lm. Furthermore, she successfully purified the PlyPSA endolysin using nickel column chromatography, verifying its purity with SDS-PAGE analysis. PD attendedUSDA Project Directors Annual Meetingfor Food Safety and Defense and Mitigating Antimicrobial Resistance Across the Food Chainon July 13, 2024 atLong Beach California. PD had the opportunity to connect with other researchers in similar fields, leading to a potentially valuable research collaboration. How have the results been disseminated to communities of interest?PD attendedUSDA Project Directors Annual Meetingfor Food Safety and Defense and Mitigating Antimicrobial Resistance Across the Food Chainon July 13, 2024 atLong Beach California. She had opportunity topresenta poster titled"Phage endolysins as disinfectant againstListeria monocytogeneson spinach" toscientific community working in related fields. What do you plan to do during the next reporting period to accomplish the goals?We have already puirified PlyPSA endolysin, we will analyze the activity of PSA against Lm on Spinatch both on room temperature and post harvesting temprature.Multiple assays will be used to verify the relative activity of theendolysin. These include the plate lysis, turbidity reduction, zymogramand the MIC. After enalyzing the activity of PSA against Lm on Spinatch wesynergy against Lm between the most active endolysins and food disinfectants at RT and on spinach at post-harvest processing temps. There are numerous food sanitizeravailable for food safety. We will choosesanitizerthat appear to be compatible (e.g. similarly active pH range) with PSA, show that the endolysin is active after exposure to the agent, and then determine the concentration of each that yields antimicrobial synergy on the spinatchusing the multiple microbicidal assays. We will proceed with purifying 6xHis-tagged PlyP511 and PlyP40 endolysins using nickel column chromatography and confirm the purification via SDS-PAGE. The purified endolysins will then undergo further analysis to assess their activity against Listeria monocytogenes (Lm) on spinach. Concurrently, we will work on expressing and analyzing LysZ5 and PlyP100 for their efficacy against Lm on spinach under similar temperature conditions.Additionally, we will evaluate the synergistic effects of endolysin-endolysin combinations and endolysin-food sanitizer combinations against Lm on spinach at both room temperature and post-harvest processing temperatures.

Impacts
What was accomplished under these goals? We have successfully expressed three 6xHis tagged endolysin proteins--Ply511, PlyP40, and PlyPSA--through IPTG induction. Activity of filter sterilized crude extracts of all three endolysins was analyzed against Lm by using multiple standard peptidoglycan hydrolase (PGH) assays, such as plate lysis, turbidity reduction, and Microbicidal assay at different pH and temperatures. PlyP40 showed the strongest inhibitory effect against Lm followed by Ply511 and PlyPSA both at 37C. PlyP40 exhibited stronger activity at pH 7 in comparison to pH 8, however, both Ply511 and PlyPSA exhibited stronger activity at pH 8 in comparison to pH 7. These crude extracts were also tested at post-harvesting temperature of 100 C. Crude extract of PlyP40 showed the strongest inhibitory effect against Lm at 100 C , however, PlySA exhibited stronger inhibitory effect against Lm in comparison to Ply511 at post harvesting temperature. We have successfully purified the 6xHis taggedPlyPSA endolysin using nickel column chromatography, verifying its purity with SDS-PAGE analysis. We are in process of analyzing the activity of purified endolysin against Lm at room and post harvesting tempratures.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Phage endolysins as disinfectant against Listeria monocytogenes on spinach Archana Sharma, Nikhat Sultana, Kynadi Heard, USDA Project Directors Meeting for Food Safety and Defense and Mitigating Antimicrobial Resistance Across the Food Chain, Annual Meeting, July 13, 2024, Long Beach California.