DEVELOPING A COMPREHENSIVE APPROACH FOR PATHOGEN CONTROL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1006126
• Project No.: ALA0SUH
• Project Start Date: Jun 1, 2015
• Project End Date: May 31, 2019

Project Director
Suh, SA, .

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Biological Sciences

Non Technical Summary
Food safety and food production are major priorities of agriculture in bothAlabama and US, and are thus important missions of the AAES. In this study, we propose toaddress both issues through a multidisciplinary approach that combines the power ofnanobiosensors for rapid and accurate detection of common foodborne pathogens withcharacterization of novel antimicrobials that can be used to combat pathogens that mayberecalcitrant to current therapy.

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

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

foodborne pathogens

rapid detection

antimicrobials

biosurfactants

genetic/metabolic engineering

Goals / Objectives
Two of the major goals of the AAES and US agriculture are to ensure a safe food supply and improve food production. The long-term goal of this project is to develop a multifaceted approach to improve control of agriculturally important pathogens. These goals are a consolidation and continuation of the research that has been ongoing in my laboratory for the past few years to improve food safety and food production. Our hypothesis is that through early detection of pathogens and development of novel antimicrobials, we can better prevent and combat foodborne illnesses and improve food production. The primary objectives of this study include 1) development of multiplex magnetoelastic nanoparticle biosensors for rapid detection of foodborne bacterial pathogens including pathogenic serovars of Salmonella enterica, Escherichia coli O157:H7, and Shigella species; 2) characterization of novel proteinaceous antimicrobials from subterranean termite Reticulotermes flavipes; 3) genetic characterization of a novel antifungal agent 7, 10, 12-trihydroxy-8(E)-octadecenoic acid produced by Pseudomonas aeruginosa; and 4) continuation of metabolic engineering of P. aeruginosa for overproduction of rhamnolipids, a biosurfactant with heat stable antifungal activity. Major expected outcomes from the proposed research include the ability of farmers and food industry personnel to easily detect contaminated foods, the development of effective antimicrobial therapy for pathogens that are resistant to existing drugs, and improved production of crops that are susceptible to fungal infections. The students working on this project will be trained in an integrated multidisciplinary approach, including microbiology, molecular biology, and biochemistry, to improve food safety and food production.

Project Methods
Research Procedures.Specific Aim 1: Development of multiplex magnetoelastic nanoparticle biosensors for rapiddetection of foodborne bacterial pathogens. In the past few years, we have optimized isolation ofhighly selective oligopeptide probes displayed on gpIII of a Ff class filamentous phage fordetection of pathogens. We currently have three manuscripts in preparation describing theseprocedures and isolation of highly selective S. Typhimurium probes. We are also in the processof improving our probes for S. Enteritidis, other pathogenic serovars of S. enterica, Shigellasonnei, and S. flexneri via limited mutagenesis to form the consensus for each target pathogen. Inaddition, we will isolate and characterize selective probes for E. coli O157:H7, S. aureus, andClostridium species. We have developed genetic approaches to construct combinatorial probesand chimeric phage probes to increase the selectivity for a given pathogen. Furthermore, we havedeveloped and demonstrated a genetic approach for stably immobilizing phage displayed probeson the magnetoelastic sensor platforms in self-assembling monolayer (SAM) to improve avidityof the sensor. We are preparing to file for a patent for this method in the near future. In order tosimultaneously detect multiple pathogens on a multiplex biosensor, each probe set for a targetorganism will be attached to a specific and different MEP of designed resonant frequency for itsdetection. MEPs ranging from 1 μm to 1000 μm in length may be used, depending upon targetagent size and mass. The corresponding characteristic resonant frequency range is from 2 GHz to2 MHz. For a MEP 50 μm long x 2 μm wide x 1 μm thick (which has a characteristic resonantfrequency of about 44 MHz), the attachment of one S. Typhimurium bacterium (mass of 1bacterium = 2 picogram) would result in a resonant frequency shift of 56 KHz, or 0.125 %. Inour previous experiments, we easily identified a 0.01% shift in resonant frequency (100 Hz outof 1 MHz) using standard instrumentation. The dimensions of the MEPs will also be adjustedbased on the properties of the target agents (principally mass and size) and the associatedfrequency change when the MEP biosensor is bound to the target. Finally, our biosensor can beeasily adapted for detection of other pathogens by coupling the MEPs with oligopeptide probesthat are specific for the infectious agents of interest. This aim will be pursued in collaborationwith Bryan Chin (Co-PI). Chin's group will be responsible for synthesis of MEPs as well as thebuilding and optimization of detection instruments. The Suh group will be responsible forisolation and development of phage probes for various bacterial pathogens. Both groups willparticipate in optimization of biosensors for detection of pathogens.Specific Aim 2: Characterization of novel proteinaceous antimicrobials from subterraneantermite Reticulitermes flavipes. We will purify and characterize each protein with antimicrobialactivity and determine its biological range especially towards multidrug resistant (MDR)bacterial pathogens. Our preliminary data indicate that R. flavipes produces agents that areeffective against several MDR pathogens. The proteins will be purified by standard approachesusing liquid chromatography. Proteins will be identified via MALDI-TOF. We are alsointerested in elucidating the changes in the antimicrobial profile of the termite when it is exposedto different pathogens. Termites only have innate immunity and yet the antimicrobial profilessuggest a specific response upon exposure to a bacterium. Once we identify the proteins, we willperform reverse genetics to identify the termite genes that encode for the antimicrobials andstudy their expressions. It is our hope that by understanding the termite immune system, we candevelop approaches to combat this important pest in the future. This aim will be pursued incollaboration with Xing Ping Hu (Co-PI).Specific Aim 3: Genetic characterization of a novel antifungal agent 7, 10, 12-trihydroxy-8(E)-octadecenoic acid produced by Pseudomonas aeruginosa. We have previously isolated severalmutants of P. aeruginosa that are defective for TOD production. We will characterize thesegenes to understand the biosynthesis of TOD in P. aeruginosa. In addition, we will determinethe biological range of TOD against a multitude of pathogens including bacteria and fungi.Finally, if successful in understanding the biosynthetic pathway of TOD, we will construct aTOD overproducing P. aeruginosa via metabolic engineering.Specific Aim 4: Continue with metabolic engineering of P. aeruginosa for overproduction ofrhamnolipids, a biosurfactant with heat stable antifungal activity. Our current metabolicallyengineered P. aeruginosa strain produces fifteen-fold more rhamnolipid than the parent strain.We will further improve the production by knocking out several more metabolic pathways thatcompete with rhamnolipid for the same precursors. In addition, we will reduce the cost ofproduction by eliminating utilization of antibiotic for maintenance of a recombinant plasmid andcostly IPTG for induction of rhamnolipid biosynthetic genes. We have developed a novel geneticapproach for maintaining plasmids without the antibiotic selection and we will implement thistechnique for this strain. In addition, we will substitute the usage of IPTG with much cheaperlactose by improving P. aeruginosa's import of the sugar and converting lactose to allolactose,the inducer of the Lac repressor. These approaches will significantly reduce the cost ofoverproducing rhamnolipids. In the future, we will use the same approach for overproduction ofTOD.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems:As explained previously, I will no longer pursue Specific Aim 2 because of the dissolution of the collaboration with Xing Ping Hu, a Co-PI. What opportunities for training and professional development has the project provided?Most of the training effort was on graduate and undergraduate students. The project outlined in Specific Aim 1 on probe isolation was pursued by threesecond year microbiology graduate students and two materials engineering graduate students. In addition, two undergraduate students contributed to this aim. All of the students were trained to be independent thinkers as well as in techniques. The students learned about the importance of foodborne pathogens as well as genetic strategies to isolate peptide probes using extensive negative andpositive selections. For the project outlined in Specific Aim 2, the senior student who pursued the study finished her researchand earned a Ph.D. in 2017. For the project described in Specific Aim 4, a M.S. student, an undergraduate, and a medical student participated in the study. How have the results been disseminated to communities of interest?The data have been disseminated through presentations at SPIE and ASM meetings and through publications inconference proceedings and in scientific journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Specific Aim 1. This past year, we made a good progress towards isolating phage probes for rapid detection ofEscherichia coliO157:H7 andCampylobacterspecies. For E. coliO157:H7 probes, after eight rounds of biopanning, we now have approximately 30 percent of the phage population in the library bind to the bacterium. We are currently in the process of identifying individual phages that bind to the bacterium via ELISA. ForCampylabacterspecies, we have taked two separate approaches. In our first approach, we took our traditional approach of isolating bacteriophage displayed oligopeptides that can bind to various species ofCampylobacterincludingC. jejuni. At present, after seven rounds of biopanning, over 50% of the phages in our library bind to a mixture of Campylobacterspecies.Using this phage library, we plan to identify individual phages that can recognize multiple species of Campylobacteras well as isolate those that are specific only forC. jejuni. In our second approach, we initated a collaboration with Dr. Robert Pantazes, a protein engineer, to synthesize those proteins (approximately 100 amino acids) based on the scaffold of fibronectin binding protein 3 (Fnb3) against uniqueC. jejuniouter membrane proteins (OMPs). During 2018, we successfully synthesized four "genes" that express these proteins and fused them to the protein III of M13 to be displayed. We are currently in the process of packaging these synthetic "phages" to test for binding toC. jejuni. Specific Aim 2. We successfully identified differentially expressed proteins in the termite hemolymph in response to pathogensPseudomonas aeruginosaandmethicillin resistantStaphylococcus aureus. We published our results in Scientific Reports. Unfortunately, due to the dissolution of collaboration with the Co-PI, Xing Ping Hu, we will no longer pursue this aim. Specific Aim 4. In our continuing efforts to bioconvert glycerol to rhamnolipids, we continued our collaborative study with Dr. Laura Silo-Suh of Mercer University School of Medicine on optimal utlization of glycerol byP. aeruginosa?. Our results were published in Microbiology.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mohamed, M. H., El-Badawy, O. H., Hadiya, S., Deaf, E. A., Suh, S.-J., Boothe, D. M., and Aly, S. A. Patterns of fluorquinolone resistance in Enterobacteriaceae isolated from the Assiut University hospitals in Egypt: a comparative study. Microb Drug Resist. 2018. Nov. 20. doi:10. 1089/mdr.2018.0249 [Epub ahead of print]
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zeng, Y., Hu, X.-P., Cao, G., and Suh, S.-J. 2018. Hemolymph protein profiles of subterranean termite Reticulitermes flavipes challenged with methicillin resistant Staphylococcus aureus or Pseudomonas aeruginosa. Scientific Reports 8: 13251.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Shuman, J., Giles, T. X., Carroll, L., Tabata, K., Powers, A., Suh, S.-J., and Silo-Suh, L. 2018. Transcriptome analysis of a Pseudomonas aeruginosa sn-glycerol-3-phosphate dehydrogenase mutant reveals a disruption in bioenergetics. Microbiology 164 (4): 551-562.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: I.-H. Chen, J. Xi, Y. Liu, S. Du, S. Horikawa, T.-S. Huang, B. A. Chin, and S.-J. Suh, Isolation of highly selective phage-displayed oligopeptide probes for detection of listeria monocytogenes in ready-to-eat food, presented at the SPIE Commercial+ Scientific Sensing and Imaging, 2018, vol. 10665, p. 106650N.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Most of the training effortwas on graduate students.The project outlined in Specific Aim 1 on probe isolation was pursued by a second year student who has abruptly left the graduate program in early 2017. Two brand new students have since picked up the project and have spent the year being trained and finally making some progress.The students learned about the importance of foodborne pathogens as well as geneticstrategies to isolate peptide probes using extensive negative and positive selections. For the projectoutlined in Specific Aim 2, the senior student who pursued the study finished her research and earned a Ph.D. in 2017. Using her experience and education, she successfully found a very good postdoctoral position and is currently pursuing that education. How have the results been disseminated to communities of interest?The data have been disseminated through presentations at SPIE, ASM, and ECS meetings and through publications in conference proceedings. In addition, we have submitted one manuscript and plan to submit second manuscript in near future. What do you plan to do during the next reporting period to accomplish the goals?My two year Hatch funding just ended in September of 2017. I plan to submit a new two year proposal in 2018 to continue the studies outlined in my five year plan.

Impacts
What was accomplished under these goals? In our second year of the Hatch project, we continued our focus on the first two Specific Aims. Specific Aim 1. A. In addition to attaching the phage molecular probes on the magnetoelastic sensor platforms via affinity interaction between streptavidin and Strep-Tag II, we also developed covalent attachment of the probes on the platform via chemical interactions. B. We are in the final stages of isolating and characterizing phage-displayed oligopeptide probes for selective binding ofEscherichia coliO157:H7 andListeria monocytogenes. We fell behind our initial schedule due to the departure of a graduate student and training of a new student. Specific Aim 2. We finished characterization of proteomes of termite hemolymphs exposed to heat-killedPseudomonas aeruginosaor methicillin resistantStaphylococcus aureus(MRSA). We performed statistical and GO analyses and verified some of the targets via RT-qPCR. We are currently in the process of submitting a manuscript describing our study to Scientific Reports.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Mohamed, M. H., El-Badawy, O. H., Hadiya, S., Deaf, E. A., Suh, S.-J., Boothe, D. W., and Aly, S. A. Patterns of fluorquinolone resistance in Enterobacteriaceae isolated from the Assiut University hospital in Egypt: a comparative study. (submitted to International Journal of Antimicrobial Chemotherapy)
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chen, I.-H., Du, S., Liu, Y., Xi, J., Lu, X., Horikawa, S., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Detection of Salmonella enterica with magnetoelastic biosensors in wash water containing Clorox and chlorine dioxide. ECS Transactions 80 (10): 1557-1564.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Du, S., Chen, I.-H., Liu, Y., Xi, J., Lu, X., Horikawa, S., Suh, S.-J., Huang, T.-S., and Chin, B.A. 2017. Biomolecular filter for the capture of bacterial pathogens in liquid streams. ECS Transactions 80 (10): 1513-1521.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Xi, J., Horikawa, S., Chen, I.-H., Du, S., Liu, Y., Lu, X., Suh, S.-J., Huang, T.-S., and Chin, B. A. 2017. Enhancement in the capture efficiency of magnetoelastic biosensors for Salmonella using a dilution method. ECS Transactions 80 (10): 1549-1555.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Horikawa, S., Du, S., Liu, Y., Lu, X., Chen, I.-H., Wikle, H. C., Chen, P., Beidaghi, M., Suh, S.-J., Feng, Y., Cheng, Z., and Chin, B. A. Effects of surface-scanning detector position on the response of a wireless magnetoelastic biosensor. ECS Transactions 80 (10): 1579-1583.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu, Y., Du, S., Horikawa, S., Chen, I.-H., Chai, Y., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Application of 2-dimensional coil detector for the sensitive direct Salmonella detection on plastic board. ECS Transactions 80 (10): 1799-1807.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu, Y., Horikawa, S., Chen, I.-H., Du, S., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Highly sensitive surfact-scannint detector for the direct bacterial detection using magnetoelastic (ME) biosensors. Proceedings for SPIE 10217, Sensing for Agriculture and Food Quality and Safety IX, 1021703.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Du, S., Chen, I.-H., Horikawa, S., Lu, X., Liu, Y., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Phage-based biomolecular filter for the capture of bacterial pathogens in liquid streams. Proceedings for SPIE 10217, Sensing for Agriculture and Food Quality and Safety IX, 1021702.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Food safety scientists and engineers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The first and foremost is in training of graduate students. The project outlined in Specific Aim 1 on probe isolation is being pursued by a new doctoral student. The student learned about the importance of foodborne pathogens as well as genetic strategies to isolate peptide probes using extensive negative and positive selections. In addition, the student learned basic microbiological and biochemical techniques necessary to isolate and characterize oligopeptide probes. For the project outlined in Specific Aim 2, the senior doctoral student working on the project had to learn to work with proteins as well as bioinformatic analyses of protein data. How have the results been disseminated to communities of interest?The data have been disseminated through presentation in SPIE meeting in April, 2016 (Specific Aim 1) and through publication in PLOS ONE (Specific Aim 2). What do you plan to do during the next reporting period to accomplish the goals?Specific Aim 1: Finish isolation and characterization of oligopeptide probes and initial testing of multiplex biosensor. Specific Aim 2: Verify the LC-MS-MS data via another round of analysis with fresh samples and validate via qRT-PCR. If funds and time permit, we will perform transcriptomics via RNAseq.

Impacts
What was accomplished under these goals? This past year, we focused on two out of four specific aims. Specific Aim 1. A. We developed a new method to make different sized magnetoelastic resonator platforms between 4 mm to 0.5 mm in length. B. We initiated isolation of peptide-displayed oligopeptide probes against two foodborne pathogens,Escherichia coliO157:H7 andListeria monocytogenes. Unfortunately, we experienced problems with both probe isolations due to the wildtype phageovertaking the phages that display oligopeptides. This problem necessitated for us to troubleshoot and develop better approaches. We are still in the process of troubleshooting this problem. Specific Aim 2.In order to characterize the antimicrobial proteins that are specifically induced in response to bacterial pathogenchallenge, we analyzed the protein profiles of isolated hemolmphs from the naive,P. aeruginosa-challenged, and MRSA-challenged termites via two independent proteomic approaches:2-dimensional gel electrophoresis andLC-MS-MS. The 2-D PAGE analyses of 493R. flavipes' hemolymph protein spots indicated that a total of 38 and 65 proteins were differentially expressed at least 2.5-fold upon being challenged withP. aeruginosaand MRSA, respectively. In contrast, LC-MS-MS analyses identified a total of 569 proteins with 49 and 29 proteins that appeared to be differentially expressed when challenged withP. aeruginosaand MRSA, respectively. Many of the proteins identified via MS analyses were related to insect metabolism, development, stress response, immune signaling, and immune effectors.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, I-Hsuan Chen, Songtao Du, Yuzhe Liu, Howard C. Wikle, Sang-Jin Suh, James M. Barbaree and Bryan A. Chin, Method for detection of a few pathogenic bacteria and determination of live versus dead cells, SPIE Proceeding Vol. 9864, 98630H, Sensing for Agriculture and Food Quality and Safety VIII, (May 2016).
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Zeng, Y., X. P. Hu, and S.-J. Suh. 2016. Characterization of antibacterial activities of eastern subterranean termite Reticulitermes flavipes against human pathogens. PLoS ONE 11(9): e0162249. doi:10.1371/journal.pone.0162249.

Progress 06/01/15 to 09/30/15

Outputs
Target Audience:Anyone involved intesting foodborne pathogens, entomologists interested in insect immunology, and microbiologists combating multidrug resistant bacterial pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the first project, a new first year graduate student was trained in basic microbiological techniques including phage isolation, enrichment, and affinity tagging. In the second project, an entomology graduate student with limited microbiological techniques was trained in basic biochemical and molecular biological techniques including protein gel electrophoresis and liquid-chromatography. How have the results been disseminated to communities of interest?We are currently finishing up a manuscript on inducible immune activity of termites for submission in January, 2016. What do you plan to do during the next reporting period to accomplish the goals?1. Isolate and characterize phage-displayed oligopeptide probes against Escherichia coli O157:H7 and Listeria monocytogenes. 2. Identify differentially expressed termite immune proteins and construct a cDNA library to pull out the genes encoding those proteins.

Impacts
What was accomplished under these goals? During this period, we focused on the first two objectives: 1) Development of multiplex magnetoelastic nanoparticle biosensors for rapid detection of foodborne bacterial pathogens; and 2) characterization of novel proteinaceous antimicrobials from subterranean termite. In the first objective, we have acquired three fluorescent markers that can be expressed in different enteric bacteria so we can visualize the selective detection of Salmonella enterica serovar Typhimurium from other S. enterica. In addtion, we started the isolation of phage-displayed peptide probes for detection of pathogenic Escherichia coli. In the second objective, we discovered that termite innate immune system can be induced with a particular bacterial pathogen to produce antibacterial proteins that are effective against the inducer bacterium. In an effort to understand and characterize the antibacterial proteins produced by the termites, we recently performed a two-dimensional gel electrophoresis and identified differentially expressed proteins.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Shin Horikawa, Yating Chai, Howard C. Wikle, Jing Dai, Jiajia Hu, Sang-Jin Suh, Vitaly Vodyanoy, and Bryan A. Chin. Nature-inspired magneto elastic biosentinels for the detection of pathogenic bacteria in stagnant liquids. Sensing for Agriculture and Food Quality and Safety VII, Proc. of SPIE.