DEVELOPING AN IMMUNOCHEMICAL FINGERPRINTING ANALYSIS FOR IDENTIFICATION OF SALMONELLA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 1004840
• Project No.: TENX-1505-FS
• Project Start Date: Dec 22, 2014
• Project End Date: Sep 30, 2017

Project Director
Chen, FU.

Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209

Performing Department
Family and Consumer Sciences

Non Technical Summary
The reduction of the level of human illness from foodborne pathogens has significant economic importance and is on the higher priority in the USDA Strategic Plan. Salmonella is one of the most important foodborne pathogens. Every year, Salmonella is estimated to cause one million illnesses in the United States. Advanced detection technology is needed to provide reliable and efficient identification of Salmonella. This project seeks to develop a new method which is reliable, fast, and economic for food industry and regulatory agencies. The proposed method is a process of subtyping Salmonella using fingerprint profiles generated from antigenic fragments. This is desirable because currently there is no such method available. Developing a fingerprint database derived from such method may serve as groundwork for further comparisons with other molecular subtyping methods. Having a reliable means of comparison can then be used to validate the developed method and to assess the relations of the method to other subtyping methods.

Animal Health Component

0%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

100%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1090	50%
712	4010	1100	50%

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

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1090 - Immunology; 1100 - Bacteriology;

Keywords

food safety

foodborne pathogens

salmonella

molecular typing

surface antigens

monoclonal antibodies

Goals / Objectives
The overall goal of this project is to develop an innovative method for identification and subtyping of Salmonella and to compare the discriminatory potential of the developed method with other molecular methods. This new method, based on profiling proteolytic fragments of Salmonella flagella antigens, is expected to be specific, rapid, and reliable. The specific objectives are to: 1. Develop analytical protocols for the Immunochemical Fingerprint Analysis (IFA). 2. Establish an IFA database of collected Salmonella serotypes. 3. Compare the relation and diversity of the IFA profiles with other molecular methods.

Project Methods
Phase 1: Development of analytical protocols for IFA The IFA method to be developed consists of four major steps, antigen extraction, limited proteolysis, SDS-polyacrylamide gel electrophoresis, and Western blot. Each of these steps needs to be standardized in order to achieve reproducible results. In general, the flagella antigens will be extracted from enriched cultures using glycine-hydrochloride or urea method and treated with various proteinases. The resulting proteolytic fragments will be separated by SDS gels and transferred to nitrocellulose membranes. Western blots will be performed using flagella-specific monoclonal antibodies that have been produced in a previous project. The antigenic fragment profiles will be captured using an imaging system and analyzed using bioinformatics software. Flagella antigen preparation: Two procedures will be tested according to the lysis buffers used. Salmonella cultures will be centrifuged at 15,000 g for 30 min, resuspended in lysis buffer and incubated for 30 min at 37 ºC. The cells will then be homogenized using a bench mixer, in order to release the flagella filaments and then centrifuged at 15,000 g for 30 min. The flagellin-containing supernatant will be submitted to ultrafiltration with a 10 kD cut-off membrane to eliminate molecules with less than 10 kD from the culture medium produced by the bacterial strain. The recovered filtrates will be used for limited proteolysis. Limited proteolysis: Trials of limited proteolysis using trypsin, Arg-C, Lys-C, Glu-C, Asp-N and Proteinase K will be carried out. The enzyme to protein ratio will be optimized to achieve time-resolved cleavage with most diverse profiles. The protein concentration will be determined by UV absorbance. Aliquots will be withdrawn from the reaction mixture at specified time intervals for electrophoresis. Gel electrophoresis: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) will be performed with a Criterion electrophoresis cell (Bio-Rad) according to manufacturer instructions. Proteins will be stained with SYPRO Ruby protein stain and protein contents will be determined by densitometry by using ChemiDoc MP imaging system and Quantity One software. Western Blot: The proteins will be transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane will be blocked with 5% non-fat dry milk powder in PBS for 1 h at room temperature and incubated for 1 h with flagella-specific monoclonal antibodies. After washing with 0.05% Tween-20 in PBS (PBS-T), bound antibodies will be reacted with goat anti-mouse IgG conjugated with peroxidase. After incubation for 1 h with the conjugate and washing with PBS-T, bound antibodies will be detected with Chemiluminescent western blot detection kit (Bio-Rad). The membrane will be photographed with illumination from a ChemiDoc MP imaging system (Bio-Rad).Phase 2: Establishment of an IFA database The IFA protocols developed in Phase 1 will be applied to establish a database of the fingerprint profiles of Salmonella enterica isolates. The genus Salmonella consists of only two species: S. enterica and S. bongori. Most Salmonella encountered in foodborne outbreaks are serotypes belonging to S. enterica which is further divided into six subspecies. The completion of IFA database will allow the comparison of fingerprint profiles of different Salmonella serotypes. The database is also an important tool for evaluating the similarity and diversity of IFA concurrently with other molecular method (PFGE) as planned in Phase 3. IFA Database of Salmonella: Salmonella enterica isolates of known serotypes will be obtained from ATCC. These isolates will consist of at least eight different serovars of Salmonella enterica subsp. enterica historically associated with foodborne outbreaks. IFA fingerprint profiles of Salmonella serovas will be established according to the protocols developed in Phase 1. Analysis of IFA Fingerprint profiles: The program BioNumerics (Applied Maths) will be used to perform numerical analyses of fingerprint profiles. Strain relationships will be inferred by use of the Pearson product-moment correlation coefficient and unweighted pair-group with mathematical average clustering, and depicted in dendrogrammatic form. Reproducibility will be determined by assessing the mean similarity between duplicate profiles of 10 randomly selected strains. Individual fingerprinting types will be defined by application of this reproducibility cut-off value to the dendrogram containing all isolates. The discriminatory potential of the method will be assessed by use of algorithm discussed by Hunter (1990), in which values between 1.0 and 0 may be obtained, whereby the value of 1.0 denotes that all strains are differentiated into individual types and the value of 0 assigns all strains studied to the same type.Phase 3: Validation of the relation and diversity between IFA and PFGE profiles This will be the first attempt to correlate the IFA fingerprint profiles with serotyping method and to compare the similarity and diversity of fingerprint profiles between IFA and PFGE. In this phase of project, a database of PFGE fingerprint profiles will be created for the known serotypes as those used in establishing the IFA database. Analysis will be conducted to compare the similarity, diversity, discriminatory potential and relation of Salmonella fingerprint profiles between IFA and PFGE. PFGE database of Salmonella: PFGE fingerprint profiles of Salmonella isolates will be created. The PFGE protocols as described by Bender et al. (2001) will be followed. In brief, the plugs will be individually digested with XbaI and the digested DNA fragments will be separated with an electrophoresis apparatus (CHEF-DR III, Bio-Rad). A strain of S. enterica serotype typhimurium will be chosen as a standard. Gels will be run with the use of 0.5× TBE buffer at 14°C, a linear increase in switching times (from 10.3 to 64.0 seconds) over a period of 22 hours, a 120-degree switch angle, and a gradient of 6.0 V per centimeter. The gels will be stained with ethidium bromide solution and photographed with ultraviolet illumination from a ChemiDoc MP imaging system. Analysis of PFGE fingerprint profiles: The gel images will be processed and analyzed by BioNumerics software. The images will be normalized by use of standard molecular markers, and banding patterns will be compared. Similarity analysis will be performed using Dice coefficients, with a 1.0% band position tolerance and 1.56% optimization, and isolates will be separated into similarity clusters by the unweighted-pair group method using average linkages. Comparison of IFA and PFGE: The random forest classification algorithm will be used to distinguish the serotypes of samples based on their IFA and PFGE profiles (Zou et al., 2010). The IFA and PFGE profiles band classes of various sizes will be generated using BioNumerics software. The band classes will be coded as 1 and 0, representing the presence and absence of a band, respectively. In the classification analysis, the profiles will be partitioned into a training set and a separate test set. The model development involved two phases: (1) building of a classification model, including determination of the classification algorithm, identification of the most relevant features (band classes), and fitting of the prediction model to training data; and (2) assessment of the performance of the prediction model. The leave-one-out cross-validation approach will be used in the analysis and to evaluate the performance of the prediction model.

Progress 12/22/14 to 09/30/17

Outputs
Target Audience:The developed method for rapid identification and subtyping of Salmonella is beneficial for the food producers/processors, and food safety laboratories. Interested industrial partners have inquired the opportunity of the new technology development. Students and faculty participated in the project have learned knowledge and skills in identification and subtyping of Salmonella. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three seminars on new development for detection of Salmonella were presented to graduate students and faculties in Agricultural Biotechnology. This project has trained one doctoral graduate student in detection and identification of Salmonella in foods using cultural and molecular methods. The student has presented the project findings in the following conferences. Devendra Bhandari. 2017. Using antigen-antibody binding kinetic parameters to characterize Salmonella enterica Serovars. Research Symposium of the Association of 1890 Research Director, Atlanta, GA, April 2, 2017 Devendra Bhandari. 2017. Salmonella detection and subtyping based on flagellar antigen-monoclonal antibody kinetics parameters. Institute of Food Technologists Volunteer Section 2017 Spring Meeting, Nashville, TN, April 28, 2017 How have the results been disseminated to communities of interest?The results from this project have been presented to food industry interested in advanced detection technologies at Institute of Food Technologies Annual Meeting and Food Expo, Las Vegas, NV, June 25-28, 2017. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project has evaluated a process of identification and subtyping of Salmonella using Immunochemical Fingerprint Analysis (IFA) profiles generated from the antigenic fragments probed by a panel of monoclonal antibodies. Four types of monoclonal antibodies capable of detecting different epitopes of Salmonella flagellar antigens were used in the studies. Western blots were performed using the four types of epitope-specific monoclonal antibodies. The antigenic fragment patterns were captured by a digital imaging system and analyzed using bioinformatics software. Both IFA (with and without endoproteinase treatment) produced fingerprints that enable the differentiation among the serovars. Endoproteinase treatments did not essentially increase the diversity of the IFA profiles. The IFA is specific and applicable as a diagnostic assay to identify and differentiate Salmonella at subspecies level. The IFA protocols have been used to establish a database of the fingerprint profiles of Salmonella isolates. The establishment of IFA database allowed the comparison of fingerprint profiles of different Salmonella serovars. We have also compared the binding kinetic parameters among Salmonella enterica serovars using the same monoclonal antibodies. The monoclonal antibody specific to flagellar antigens was immobilized on a dextran matrix attached to the Surface Plasmon Resonance sensor surface. The interaction of the antibody with Salmonella flagellar antigens in solution was monitored in real time. Control of the amount of immobilized ligand and reusable sensor surfaces permitted the comparison of f flagellar antigens from various Salmonella serovars reacting with monoclonal antibodies under almost identical conditions. The results showed that there were five distinct patterns in binding kinetics among serovars; however, the binding kinetics analysis does not necessarily correspond to serotypes. This project presented the potential of using anti- flagellar antigens monoclonal antibodies as recognition elements in the subtyping and rapid detection of Salmonella.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chen, F.C., and Bhandari, D. 2017. Detection and characterization of Salmonella based on kinetics of monoclonal antibody-antigen reactions, Institute of Food Technologists Annual Meeting, Las Vegas, June 26, 2017

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

Outputs
Target Audience:The detection method is intended to be used by regulatory agencies, meat and poultry producers/processors to facilitate identification of dangerous foodborne pathogens in the processing facilities and final products. Interested industrial partners have inquired the opportunity of the new technology developments. Students and faculty participated in the project have learned knowledge and skills in identification and subtyping of Salmonella. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained one graduate student in detection and identification of Salmonella in foods using cultural and molecular methods. The student has presented the project findings at 38th Annual University-Wide Research Symposium, Tennessee State University, April 4 -8, 2016 How have the results been disseminated to communities of interest?The results from this project have been communicated with food industry interested in advanced detection technologies during the Institute of Food Technologists Annual Meeting in Chicago, July 16-19, 2016 What do you plan to do during the next reporting period to accomplish the goals?This project will conduct further experiments to evaluate the similarity and diversity of IFA concurrently with other molecular methods. The IFA database will be used to compare the relation and diversity of the profiles with other molecular methods.

Impacts
What was accomplished under these goals? Analytical protocols for the Immunochemical Fingerprint Analysis (IFA) was developed and tested on Salmonella enterica serovars. The IFA method developed consists of three major steps including limited proteolysis, 2-D/SDS-polyacrylamide gel electrophoresis, and Western blot. In brief, the flagellar antigens were extracted from cultures using glycine-hydrochloride and treated with various protenases (Trypsin, Lys-C, Glu C, Asp-N and Arg-C). The resulting proteolytic fragments were separated by 2-D/SDS gels and transferred to nitrocellulose membranes. Western blots were performed using a cocktail of flagella-specific monoclonal antibodies. The antigenic fragment patterns were captured by a digital imaging system and analyzed using bioinformatics software. The resulting proteolytic fragments yielded more than 15 detectable peptides with molecular mass ranging from 52 kD to 16 kD and isoelectric point ranging from 4.6 to 5.1. Comparisons of the fingerprint heterogeneity generated from limited proteolysis enable the differentiation of Salmonella serotypes. The developed IFA method is both specific and sensitive and thus could be valuable as a diagnostic assay to identify and differentiate Salmonella at subspecies levels. We have also analyzed flagellar antigen using a panel of monoclonal antibodies and compared the binding kinetic parameters among Salmonella enterica serovars. Monoclonal antibody specific to flagellin was immobilized on a dextran matrix attached to the SPR sensor surface. The interaction of the antibody with Salmonella flagellin in solution was monitored in real time. Control of the amount of immobilized ligand and reusable sensor surfaces permitted the comparison of flagellin from various Salmonella serovars reacting with monoclonal antibodies under almost identical conditions. Differences in affinity and reaction rates were derived from the association and dissociation curves. The calculated association rate constants (Ka) for flagellin from ten Salmonella strains ranged from 9.8 × 104 to 1.5 × 107 M−1 s−1 and the dissociation rate constants (Kd) ranged from 2.3 × 10−4 to 6.2 × 10−2 s−1. The assay sensitivity to flagellin was determined between 0.31 and 9.8 nM. Cluster analysis of Ka and Kd correlated directly with the antigenic properties of the corresponding Salmonella serovars. This study showed the potential of using anti-flagellin monoclonal antibodies as recognition elements in the SPR biosensor for subtyping and rapid detection of Salmonella. This detection method could help identify the contaminated food sources and thereby prevent foodborne diseases.

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: Chen, F.C., Zhou, S., Nahashon, S., and Bridgman, R.C. 2016. Detection of Salmonella and Campylobacter in chicken rinse water using a surface plasmon resonance sensor. In Microbes in the spotlight: recent progress in the understanding of beneficial and harmful microorganisms (pp.186-190). A. Mendez-Vilas, Ed, BrownWalker Press, Barcelona, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Chen, F.C., and Bhandari, D. 2016. Immunochemical Fingerprint Analysis for Identification and Subtyping of Salmonella, Institute of Food Technologists Annual Meeting, Chicago, July 19, 2016

Progress 12/22/14 to 09/30/15

Outputs
Target Audience:The developed method is intended to be used by regulatory agencies, meat and poultry producers/processors to facilitate identification of dangerous foodborne pathogens in the processing facilities and final products. Interested industrial partners have inquired the opportunity of the new technology developments. Students and faculty participated in the project have learned knowledge and skills in identification and subtyping of Salmonella. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student was trained in detection and identification of Salmonella in foods using cultural and molecular methods. How have the results been disseminated to communities of interest?The PD has communicated the new development of the IFA with food safety professionals and testing laboratories. What do you plan to do during the next reporting period to accomplish the goals?The IFA protocols developed will be applied to establish a database of the fingerprint profiles of Salmonella isolates. The establishment of IFA database will allow the comparison of fingerprint profiles of different Salmonella serotypes. The database is also an important tool for evaluating the similarity and diversity of IFA concurrently with other molecular methods. The results from this project will be communicated with food industry interested in advanced detection technologies.

Impacts
What was accomplished under these goals? Experiments were conducted to study Salmonella flagellar antigens using a panel of seven monoclonal antibodies. Antigen specificity of monoclonal antibodies was characterized by enzyme-linked immunosorbent assay and 2-D gel electrophoresis. These monoclonal antibodies specific to flagella antigens were applied to immunochemical fingerprint analysis (IFA) for identification and subtyping of Salmonella. The method developed consists of three major steps including limited proteolysis, SDS-polyacrylamide gel electrophoresis, and Western blot. In summary, the flagella antigens were extracted from enriched cultures using glycine-hydrochloride and urea methods and treated with various proteinases. The resulting proteolytic fragments were separated by SDS gels and transferred to nitrocellulose membranes. Western blots were performed using flagella-specific monoclonal antibodies. The antigenic fragment profiles were captured using an imaging system and analyzed using bioinformatics software. Standardized laboratory protocols were prepared for training graduate students in performing limited proteolysis, SDS-polyacrylamide gel electrophoresis, Western blot, and image analysis.

Publications