Source: PURDUE UNIVERSITY submitted to
HIGH THROUGHPUT, RAPID, FOODBORNE PATHOGEN SCREENING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1000028
Grant No.
(N/A)
Project No.
IND011634
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Project Director
Bhunia, AR, K.
Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Food Science
Non Technical Summary
Foodborne diseases are responsible for approximately 48 million illnesses with 3000 deaths annually in the US (Scallan et al., 2011). There are over 200 known microbial, chemical or physical agents that can result in illness when consumed (Newell et al., 2010). Of these, microbial source comprising of bacterial, viral and fungal are of major concern. CDC estimates that of all the foodborne infections, 44% of the hospitalizations and deaths are attributed to 31 known pathogens (Scallan et al., 2011). Among these pathogens, the majority of the illnesses, hospitalizations and deaths are caused by five known pathogens, which include Norovirus, nontyphoidal Salmonella, Clostridium perfringens, Campylobacter spp. and Staphylococcus aureus. Salmonella enterica causes gastroenteritis in humans and nearly 1 million people in the US are infected annually, resulting in approximately 19,000 hospitalizations and over 378 deaths (Scallan et al., 2011).
Animal Health Component
0%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7123260110020%
7123270110020%
7123320110020%
7123450110020%
7124010104020%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3320 - Meat, beef cattle; 3260 - Poultry meat; 4010 - Bacteria; 3270 - Eggs; 3450 - Milk;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;
Goals / Objectives
The overarching objective is to develop and optimize biosensor- based assays for sensitive detection of multiple foodborne pathogens from food. The specific aims are: AIM 1. To optimize and improve sample preparation step prior to biosensor-based detection AIM 2. Improve biosensor performance by employing novel probes, reagents and media AIM 3. Optimize biosensor performance for pathogens
Project Methods
For biosensor technologies to work effectively with varieties of food matrices, sample preparation strategy and sample enrichment steps must be well defined. For culturing multiple pathogens in the same media, we have developed a multipathogen enrichment broth (SEL; Salmonella, E. coli, Listeria) that supports growth of these three pathogens simultaneously. We will determine various antibodies and mammalian cell receptors that are used by pathogens during infection as potential ligands for pathogen capture on biosensor platforms. As we have demonstrated earlier, mammalian Hsp60 protein, a receptor for L. monocytogenes LAP (Listeria adhesion protein) worked very well on a fiber optic sensor (Koo et al., 2011) and on a microfluidic biochip platform (Koo et al., 2009) for direct detection of this pathogen. Application of other pathogen-specific receptor molecules including translocated intimin receptor (TIR) for capture of Shiga-toxin producing E. coli (STEC) strains, and β1-Integrin for capture of Yersinia, Shigella and Salmonella, etc. will be studied in this project. For cloning of TIR, the gene will be PCR amplified from E. coli EDL933 (O157:H7) and inserted into pET-SUMO vector (Invitrogen) for protein expression (Jagadeesan et al., 2011). His-tagged proteins will be purified using Ni-affinity column. The optical fibers coated with antibody or receptor is first exposed to sample containing target pathogens for capture. Subsequently, pathogen-specific second antibody/aptamer/receptor conjugated to a fluorophor (Cy5, Alexa Fluor) will be added for pathogen-specific signal. The formation of pathogen-antibody/receptor sandwich will emit fluorescence that generates evanescent wave, which is detected by a built-in laser detector (Analyte 2000, Research International, Monroe, WA). Previously, we have developed fiber optic-based assays for each L. monocytogenes (Geng et al., 2004; Mendonca et al., 2012; Nanduri et al., 2006; Ohk et al., 2010), E. coli O157:H7 (Geng et al., 2006) and Salmonella enterica serovar Enteritidis (Valadez et al., 2009) and validated the assays with food products.

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:Students and food safety and food microbiology professionals Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Through peer-reviewed journal articles, conference presentations, and public speaking through invited talks What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In order to improve methods for foodborne pathogen detection, in the past five years, our team focused on target pathogen separation from complex food matrices by employing a pathogen enrichment device (PED) and antibody-coated paramagnetic beads. Both PED and paramagnetic bead separated target pathogens were efficiently detected by the biosensor, lateral flow immunoassay, and PCR methods thus providing improved specificity, sensitivity, speed and accuracy of the assays. Three major biosensor platforms were developed and validated, (i) fiber optic immunosensor, (ii) light scattering sensor and (iii) mammalian cell-based sensor. Fiber optic sensor requires specific biorecognition molecules such as antibody or aptamer and was developed for detection of Listeria monocytogenes, Salmonella enterica and E. coli O157:H7. The laser-based light scattering sensor also known as BARDOT (BActerial Rapid Detection using Optical scattering Technology) operates independent of any biorecognition molecule, directly interrogates bacterial colonies on agar plate based on their colony scattering signatures. BARDOT was successfully used for detection of multiple pathogens including Listeria monocytogenes, Shiga-toxin producing E. coli, Salmonella enterica, Bacillus spp., Staphylococcus aureus, Vibrio vulnificus, and V. parahaemolyticus and several bacteria from Enterobacteriaceae family. BARDOT was also proved to be a powerful tool for studying changes in bacterial physiology, morphology and gene deletion. Mammalian cell-based sensor, on the other hand, interrogates pathogen interaction with the host cells and is highly sensitive for the detection of low amounts of harmful target analytes. In addition, we also developed methods to control pathogens in biofilms using antimicrobial coated nanoparticles on food products or food contact surfaces.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Drolia, R., Tenguria, S., Durkes, A.C., Turner, J.R. and Bhunia, A.K. 2018. Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation. Cell Host & Microbe 23(4): 470-480.e7.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Alsulami, T.S., Zhu, X., Abdelhaseib, M.U., Singh, A.K., and Bhunia, A.K. 2018. Rapid detection and differentiation of Staphylococcus colonies using an optical scattering technology. Anal. Bioanal. Chem. 410 (22): 54455454.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 4. Zhu, X., Liu, D., Singh, A.K., Drolia, R., Bai, X., Tenguria, S., and Bhunia, A.K. 2018. Tunicamycin mediated inhibition of wall teichoic acid affect Staphylococcus aureus and Listeria monocytogenes cell morphology, biofilm formation and virulence. Front. Microbiol. 9:1352.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Singh, A.K., Bai, X., Amalaradjou. M.A.R., and Bhunia, A.K. 2018. Antilisterial and antibiofilm activity of peptide functionalized gold nanoparticles Front. Sust. Food Syst. 2:74.


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

Outputs
Target Audience:Food scientists and food microbiologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Through publications in peer-reviewed journals,and presentations at NationalConferences What do you plan to do during the next reporting period to accomplish the goals?Continue further improvement and data acquisition

Impacts
What was accomplished under these goals? AIM 1. To optimize and improve sample preparation step prior to biosensor-based detection No reportable progress AIM 2. Improve biosensor performance by employing novel probes, reagents and media (i) Characterization of anti-Listeria antibody Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. Here, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus. AIM 3. Optimize biosensor performance for pathogens To employ an optical scattering technology for rapid detection and differentiation of Staphylococcus colonies Staphylococcus species are a major human and veterinary pathogen responsible for nosocomial infections and foodborne illnesses. Staphylococcus genus includes over 30 species and subspecies, which are Gram-positive, non-motile, non- spore forming, and non-flagellate bacteria. We investigated if BARDOT (bacterial rapid detection using optical scattering technology) could be used for rapid screening and detection of colonies of Staphylococcus at the genus level on agar plates, and differentiate these from non-Staphylococcus bacteria. Among the six growth media tested, phenol red mannitol agar was found suitable for building the Staphylococcus species scatter image library as it generated maximum differentiating scatter pattern. Scatter image library for Staphylococcus species gave a high positive predictive value (PPV: 87.5%-100%) when tested against the known laboratory strains of Staphylococcus spp., while the PPV against non-Staphylococcus spp. was, 0%-38%. Total nine samples of bovine raw milk and ready-to-eat chicken salad were tested in this study, and BARDOT detected Staphylococcus with 80-100% PPV in naturally contaminated samples. A total of 45 natural isolates were further confirmed by tuf gene-specific PCR, 16S rRNA gene sequencing (1245-1409 bp) and analytical profile index (Staph-APITM) based biochemical tests. BARDOT-based identification of isolates resulted in 6.6% false-positive. This novel, label-free, non-invasive on-plate colony screening technology can be adapted by the food industries, biotechnology companies and public health laboratories for the screening of Staphylococcus from various samples to aid in food safety and public health management. To optimize biosensor performance for detection of Enterobacteriaceae from food The Enterobacteriaceae (EB) family and its most well-known members, coliforms, are used as "indicators" of hygiene monitoring, sanitization practices, and process verification of food products. BARDOT generates differential scatter pattern for a bacterial species when grown on different agar media. In this study, our goal was to find suitable agar media for identification of bacterial genera in Enterobacteriaceae family that could be used as indicator for food process verification and monitoring sanitary or hygienic practices employed during food production and manufacturing. Among the five agar media specific for Enterobacteriaceae family tested, Rapid Enterobacteriaceae (REB), Modified Kingler Iron Agar (mKIA), Glucose Bromocresol Purple Agar (GBPA), Violet Red Bile Glucose Agar (VRBGA), and MacConkey Agar (MCA), VRBGA and REB showed very high classification accuracy (>91%) when tested against 12 different genera. Though other EB media, such as VRBGA were more selective, the members of the Enterobacteriaceae family produced high amounts of chromogens that interfered with laser propagation through colonies. Therefore, REB was used to build the EB genera scatter image library. The EB scatter pattern library contains approximately 7,000 patterns belonging to the top 15 medically important EB genera. The EB scatter image library was used for the screening and detection of artificially inoculated whole milk, egg whites, and dry infant formula, and identification of natural EB isolates from bovine raw milk and slaughter house environmental samples. In parallel, ISO and 3M methods were used as verification methods. A total 30 natural isolates were further confirmed by MALDI-TOF and 16S rRNA gene sequencing. When BARDOT libraries (Lib-1, coliform group; Lib-2, major pathogens; Lib-3, miscellaneous/ opportunistic pathogens) were matched against spiked food samples with mixed cultures, variably successful identification was observed. Positive predictive values (PPV) ranged from 33% to 96.5%. Subsequent tests involved individual culture analysis where PPV values ranged from 83-100%. A positive result was established for each isolate and a BARDOT ID was obtained; however, subsequent MALDI-TOF and sequencing results confirmed these isolates were different than what BARDOT identified them as. In the same line, isolate analysis with individual genus libraries was not in agreement with the results obtained with confirmatory tests. The results overall indicate that BARDOT may accurately identify a single microorganism. However, the overlapping scatter pattern signature among EB genera may affect accuracy of identification.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Sarkar, P., Bhunia, A.K., and Yao, Y. 2017. Impact of starch-based emulsions on the antibacterial efficacies of nisin and thymol in cantaloupe juice, Food Chem. 217:155-162.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Sarkar, P., Bhunia, A.K., Yao, Y. 2016. Emulsion Stabilized with Starch Octenyl Succinate Prolongs Nisin Activity against Listeria monocytogenes in a Cantaloupe Juice Model. J. Food Sci. 81: M2982-M2987.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Kim, H., Rajwa, B., Bhunia, A.K., Robinson, J.P., and Bae, E. 2017. Development of a multispectral light scatter sensor for bacterial colonies. J. Biophoton. 10: 634-644.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Wu, X., Wei, P-H., Zhu, X., Wirth, M.J., Bhunia, A.K., and Narsimhan, G. 2017. Effect of immobilization on the antimicrobial activity of a cysteine-terminated antimicrobial Peptide Cecropin P1 tethered to silica nanoparticle against E. coli O157:H7 EDL933. Colloids and Surfaces B: Biointerfaces, 156: 305312.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fu, Y., Deering, A.J., Bhunia, A.K. and Yao, Y. 2017. Biofilm of Escherichia coli O157: H7 on cantaloupe surface is resistant to lauroyl arginate ethyl and sodium hypochlorite. Int. J. Food Microbiol. 260, 11-16.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Xiang, N., Lyu, Y., Zhu, X., Bhunia, A.K. and Narsimhan, G. 2017. Effect of physicochemical properties of peptides from soy protein on their antimicrobial activity. Peptides 94, 10-18.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fu, Y., Deering, A., Bhunia, A.K., and Yao, Y. 2017. Pathogen biofilm formation on cantaloupe surface and its impact on the antibacterial effect of lauroyl arginate ethyl. Food Microbiol. 64: 139-144.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Ryan, V. and Bhunia, A.K. 2017. Mitigation of foodborne illnesses by probiotics. In Foodborne Pathogens: Virulence Factors and Host Susceptibility. Edited by Joshua Gurtler Michael Doyle, and Jeffrey Kornacki, Springer, New York, NY. pp 603-634.


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

Outputs
Target Audience:Food safety educators, students and professionals Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and visiting scientists received traing on biosensor based methods for foodborne pathogen detection How have the results been disseminated to communities of interest?Through peer-reviewed research publications and conference presentations What do you plan to do during the next reporting period to accomplish the goals?Optimize BARDOT, Cell based sesnor and Plasmonic ELISA based biosensor methods for testing of Enterobacteriaceae and other foodborne pathogens from naturally contaminated samples.

Impacts
What was accomplished under these goals? We investigated application potential of BARDOT for detection of microbial pathogens and indicator bacteria as a tool for monitoring product safety and public health. We used SEL agar medium for simultaneous growth and detection of Salmonella, E. coli, and L. monocytogenes using BARDOT. We also screened different media for screening Enterobacteriaceae using BARDOT and Rapid' Enterobacteriaceae (REB) medium was found to be optimal, and a scatter image library was built that consist of 14 genera and 31 species. BARDOT was employed as a companion tool with fiber optics sensor for detection of Salmonella from naturally contaminated chicken carcass rinse obtained from poultry processing plants. BARDOT was also successfully used to monitor the effects of antibiotic resistance on the scatter patterns of Salmonella, and mutation in virulence genes in L. monocytogenes. In addition, we have demonstrated that Listeria adhesion protein (LAP), can be used as a bio-recognition molecule for detection of L. monocytogenes and L. ivanovii upon immobilization on magnetic beads for specific capture from food samples.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Singh, A.K., Leprun, L., Drolia, R., Bai, X., Kim, H., Aroonnual, A., Bae, E., Mishra, K.M., and Bhunia, A.K., 2016. Virulence gene-associated mutant bacterial colonies generate differentiating 2-D laser scatter fingerprints. Appl. Environ. Microbiol. 82:3256-3268.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Singh, A.K. and Bhunia, A.K. 2016. Optical scatter patterns facilitate rapid differentiation of Enterobacteriaceae on CHROMagarTM Orientation medium. Microbial Biotechnology 9(1):127-135.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Abdalhaseib, M.U., Singh, A.K., Bailey, M., Singh, M., and Bhunia, A.K. 2016. Fiber optic and light scattering sensors: complimentary approaches to rapid detection of Salmonella enterica in food samples. Food Control 61:135-145.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Bi, L., Yang, L., Bhunia, A.K. and Yao, Y. 2016. Emulsion stabilized with phytoglycogen octenyl succinate prolongs the antimicrobial efficacy of ?-poly-L-lysine against Escherichia coli O157:H7. LWT - Food Science and Technology. 70:245-251
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wu, X, Singh, A.K., Wu, X., Lyu, Y., Bhunia, A.K., and Narsimhan, G. 2016. Characterization of antimicrobial activity against Listeria and cytotoxicity of native melittin and its mutant variants. Colloids and Surfaces B: Biointerfaces. 143:194-205.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mendon�a, M., Moreira, G.M.S.G., Concei��o, F.R., Hust, M., Mendon�a, K.S., Moreira, A.N., Fran�a, R.C., da Silva, W.P., Bhunia, A.K., Aleixo, J.A.G. 2016. Fructose 1,6-bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria species. PLoS ONE 11(8): e0160544
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang, D., Coronel-Aguilera, C.P., Romero, P.L., Perry, L., Minocha, U., Rosenfield, C., Andrew G. Gehring, A.G., Paoli, G.C., Bhunia, A.K., and Applegate, B. 2016. The Use of a Novel nanoLuc-based reporter phage for the detection of Escherichia coli O157:H7. Sci. Rep. 6, Article number: 33235
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xiang, N., Lyu, Y., Zhu, X., Bhunia, A.K., and Narsimhan, G. 2016. Methodology for Identification of Pore Forming Antimicrobial Peptides from Soy Protein Subunits ?-conglycinin and Glycinin Peptides. Peptides 85:27-40
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Fu, Y., Bhunia, A.K. Sarkar, P. and Yao, Y. 2016. Delivery Systems of Antimicrobial Compounds to Food. Trends Food Sci. Technol. 57:165-177.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kim, H., Doha, I-J., Sturgis, J., Bhunia, A.K., Robinson, J.P., and Bae, E. 2016. Reflected scatterometry for non-invasive interrogation of bacterial colonies. J. Biomed. Opt. 21(10): 107004.
  • Type: Books Status: Published Year Published: 2016 Citation: Sensors for Food Safety and Quality. 2016. Editor, A.K. Bhunia, MDPI, Sensors, ISSN 1424-8220, Pages 220.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Singh, A.K., Amalaradjou, M.A.R., and Bhunia, A.K. 2016.Listeria adhesion protein augments antilisterial and antibiofilm activity of peptide functionalized gold nanoparticles. 2016-A-5175-Microbe; ASM Annual Meeting, Boston, MA, June 16-20
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Martinez, M., Singh, A.K., Bhunia, A.K., 2016. Light-scattering sensor for detection and identification of indicator bacteria Enterobacteriaceae family for process verification and hygiene monitoring. E17-003 (e-poster), IFT annual meeting, Chicago, IL. July16-19, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ryan, V.E., Bailey, T.W., Vemulapalli, T., Durkes, A., and Bhunia, A.K. 2016. Health beneficial attributes of bioengineered probiotics against subclinical infection of Listeria monocytogenes in pregnant guinea pigs. E20-003 (e-poster), IFT annual meeting, Chicago, IL. July16-19, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Wu, X., Zhu, X., Wei, P., Wirth, M., Bhunia, A.K., and Narshimhan, G. 2016. P02-061, IFT Annual Meeting, Chicago, IL. July16-19, 2016
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bonilla, J., Ryan, V., Bhunia, A.K., and Kokini, J., 2016. Specific anti-HMW glutenin and anti-LMW glutenin antibodies development by comparative genomics. P05-007. IFT Annual Meeting, Chicago, IL. July16-19, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bai, X., Koo, O.K, and Bhunia, A.K. 2016. Mammalian cell-based in vitro pathogenicity analysis of Listeria monocytogenes biofilm-forming cells. T6-11 (Technical oral presentation); IAFP Annual Meeting, St. Louis, MO, July 31  Aug 3, 2016.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Chen, J., Tang, J., Bhunia, A.K., Tang, C., Wang, C., Shi, H. 2015. Development of a multi-pathogen enrichment broth for simultaneous growth of five common foodborne pathogens. J. Gen. Appl. Microbiol. 61(6): 224-231.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Food scientists and students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?1. Through peer-reviewed publications 2. Conference presentation 3. Class-room instructions What do you plan to do during the next reporting period to accomplish the goals?1. Continue biosensor based method development for other foodborne pathogens including Staphylococcus spp., Yersinia enterocolitica and Enterobacteriaceae family. 2. Continue testing biosensor (BARDOT) with naturally contaminated food samples

Impacts
What was accomplished under these goals? AIM 1. To optimize and improve sample preparation step prior to biosensor-based detection Pathogen Enrichment Device (PED): The bottleneck for accurate detection of foodborne pathogens is separation of target analytes from complex food matrices. Currently used sample preparation methods are cumbersome, arduous and lengthy; thus, a user-friendly system is desirable. A hand-held sample preparation system designated pathogen enrichment device (PED) was built that contains a growth chamber, filters, and an ion exchange cartridge to deliver bacteria directly onto the detection platforms. Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes were used as model pathogens. Spinach, ground beef, hotdogs, and eggs were used as model foods to evaluate PED performance, and results were compared with traditional bag enrichment method. Bacterial cells were inoculated at 1, 10, 100 CFU/g of sample and enriched in PED using appropriate pathogen-specific selective enrichment broths. The bacterial cell counts in both PED and stomacher-bag were comparable and the pH in PED-recovered cell suspension was close to neutral whereas the pH of cell suspension in the stomacher-bag was slightly acidic. The bacterial recovery from the PED was 95-100% and were directly detected by lateral-flow immunoassay (LFIA), quantitative PCR (qPCR) and light scattering sensor with sample-to-result time of 8-24 h with a detection limit of 1 CFU/g. In qPCR, the amplified PCR products appeared in 4-5 cycles earlier with PED-enriched cultures compared to the cultures enriched in stomacher-bag. The hand-held PED proved to be a one-step procedure for enrichment and recovery of homogenous particle-free bacterial cells for detection using immunological, molecular or biosensor-based platforms. AIM 2. Improve biosensor performance by employing novel probes, reagents and media We investigated streptomycin-induced stress response in Salmonella enterica serovars Typhimurium using BARDOT by monitoring changes in the colony scatter patterns. Streptomycin was used as a model antibiotic since Salmonella spp. are generally resistant and show high tolerance for this antibiotic. BARDOT revealed antibiotic induced differential scatter patterns which is attributed to the expression of bacterial chaperonin GroEL, identified by mass spectrometry (MALDI-TOF). GroEL is a stress response protein whose expression was further verified by qPCR, ELISA and Western blot. This study demonstrates BARDOT's potential as a research tool to study antibiotic response in bacteria. AIM 3. Optimize biosensor performance for pathogens Detection of Salmonella from chicken carcass rinse: Antibodies based fiber optic sensor was developed and integrated with light scattering sensor for Salmonella enterica detection. Total 50 chicken carcass rinse samples were tested for the presence of Salmonella enterica using these methods. Out of 50 only 4 samples (8%) were found contaminated with Salmonella enterica. BARDOT identified Salmonella positive samples with 100% accuracy after matching with the Salmonella and non-Salmonella bacteria scatter image libraries. Detection of Enterobacteriaceae: BARDOT generates differential scatter pattern for a bacterial species when grown on different agar media. In this study, our goal was to find suitable agar media for identification of bacterial genera in Enterobacteriaceae family that could be used as indicator bacteria for food process verification and monitoring sanitary or hygienic practices employed during food production and manufacturing. Among the five agar media specific for Enterobacteriaceae family tested, Rapid Enterobacteriaceae (REB), Modified Kingler Iron Agar (MKIA), Glucose Bromocresol Purple Agar (GBPA), Violet Red Bile Glucose Agar (VRBGA), and MacConkey Agar (MCA), VRBGA and REB showed very high classification accuracy (>97%) when tested against 12 different genera.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, A.K. and Bhunia, A.K. 2015. Optical scatter patterns facilitate rapid differentiation of Enterobacteriaceae on CHROMagarTM Orientation medium. Microbial Biotechnology. 10(doi:10.1111/1751-7915.12323)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, A.K., Drolia, R., Bai, X., and Bhunia, A.K. 2015. Streptomycin induced stress response in Salmonella enterica serovar Typhimurium shows distinct colony scatter signature. PLoS ONE 10(8): e0135035
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, A.K., Sun, X., Bai, X., Kim, H., Abdalhaseib, M.U., Bae, E., and Bhunia, A.K. 2015. Label-free, non-invasive light scattering sensor for screening of Bacillus colonies. Journal of Microbiological Methods 109:56-66
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kim, K.-P., Singh, A.K., Bai, X., Leprun, L., and Bhunia, A.K. 2015. Novel PCR assays complement laser biosensor-based method and facilitate Listeria species detection from food. Sensors (Basel) 15:22672-22691
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hahm, B.-K., Kim, H., Singh, A.K., and Bhunia, A.K. 2015. Pathogen enrichment device (PED) enables, one-step growth, enrichment and separation of pathogen from food matrices for detection using bioanalytical platforms. Journal of Microbiological Methods 117:64-73
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Qiy, Y.-L., He, Q.-H., Xu, Y., Bhunia, A.K., Tu, Z., Chen, B., Liu, Y.-Y. 2015. Deoxynivalenol-mimic nanobody isolated from a na�ve phage display nanobody library and its application in immunoassay. Analytica Chimica Acta 887:201-208
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: He, Y., Reed, S., Bhunia, A.K., Gehring, A., Nguyen, L.-H., and Irwin, P.L. 2015. Rapid identification and classification of Campylobacter spp. using laser optical scattering technology, Food Microbiology 47:28-35
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Singh, A.K., Sun, X., Bai, X., Kim, H., Abdalhaseib, M., Bae, E., and Bhunia, A.K. 2015. Label-free, non-invasive light scattering sensor for screening of Bacillus colonies. Journal of Microbiological Methods 109:56-66
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Josefsen, M. H., Bhunia, A.K., Olsson Engvall, E., S�ndergaard, M. S. R. and Hoorfar, J. 2015. Monitoring Campylobacter in the poultry production chainFrom culture to genes and beyond. Journal of Microbiological Methods 112:118-125
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kim, H., Doh, I-J., Bhunia, A.K., King, G.B., and Bae, E. 2015. Scalar diffraction modeling of multispectral forward scatter patterns from bacterial colonies. Optics Express 23 (7):8545-8554
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ansari, S., Bozkurt, F., Yazar, G., Ryan, V., Bhunia, A. and Kokini, J. 2015. Probing the distribution of gliadin proteins in dough and baked bread using conjugated quantum dots as a labeling tool. Journal of Cereal Science 63:41-48
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Il-Hoon Cho, I-H., Bhunia, A.K., Irudayaraj, J. 2015. Rapid pathogen detection by lateral-flow immunochromatographic assay with gold nanoparticle-assisted enzyme signal amplification. International Journal of Food Microbiology 206:60-66
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Singh, A.K., Sarkar, P., Janaswamy, S., Yao, Y., and Bhunia, A.K. 2014. Encapsulation and Delivery of Antimicrobial Compounds, In Novel Food Preservation and Microbial Assessment Techniques, Chapter 8. Editor, Ioannis S. Boziaris, CRC Press, Boca Raton, FL. Pp. 218-236.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Bhunia, A.K., Taitt, C.R., and Kim, M.S. 2015. High throughput screening strategies and technology platforms for detection of pathogens in food: An Introduction, In High Throughput Screening for Food Safety Assessment: Biosensor Technologies, Hyperspectral Imaging and Practical Applications. Chapter 1, Editors, A. K. Bhunia, M.S. Kim, and C.R. Taitt. Woodhead Publishing, Cambridge, UK. Pp. 1-9.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Bae, E. and Bhunia, A.K. 2015. Label-free light scattering sensors for high throughput screening of microbes and toxins in food. In High Throughput Screening for Food Safety Assessment: Biosensor Technologies, Hyperspectral Imaging and Practical Applications. Chapter 6, Editors, A.K. Bhunia, M.S. Kim, and C.R. Taitt. Woodhead Publishing, Cambridge, UK. Pp. 149-162.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Mendonca, M., and Bhunia, A.K. 2015. Fiber Optic Sensors for High Throughput Screening of Pathogens, In High Throughput Screening for Food Safety Assessment: Biosensor Technologies, Hyperspectral Imaging and Practical Applications. Chapter 14, Editors, A.K. Bhunia, M.S. Kim, and C.R. Taitt. Woodhead Publishing, Cambridge, UK. Pp. 249-259.
  • Type: Books Status: Published Year Published: 2015 Citation: High Throughput Screening for Food Safety Assessment: Biosensor Technologies, Hyperspectral Imaging and Practical Applications. 2015. Editors: A. K. Bhunia, Moon S. Kim, and C.R. Taitt. Woodhead Publishing, Cambridge, UK. Pages 523
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Singh, A.K., Bai, X., Leprun, Tang, Z., L., Drolia, R., Kim, H., Aroonual, A., Bae, E., Mishra, K.K., Bhunia, A.K. 2015. Laser scatterometer: a novel optical sensor for real-time, non-invasive rapid screening of mutant Listeria monocytogenes colonies. In Institute of Food Technologists 16th General Meeting. July 11-14, 2015, Chicago, IL. Abstract #11803.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Wu, X., Narsimhan, Singh, A, and Bhunia, A. 2015. Antimicrobial activity of wild type and mutant melitin against Listeria monocytogenes. IFT annual meeting, Chicago, IL. July 11-14, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Xiang, N., Narsimhan, Lyv, Y., and Bhunia, A. 2015. Antimicrobial peptide segments from soy protein for use in food safety. IFT annual meeting, Chicago, IL. July11-14, 2015
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Bailey, M., Brar, J., Corkran, S.,Ebner, P., Oliver, H., Bhunia, A., and Singh, M. 2015. Prevalence and antimicrobial resistance of Salmonella during conventional and organic processing of antibiotic-free broilers. IAFP annual meeting. Technical Talk (T11-06), Portland, OR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Brar, J., Bailey, M., Corkran, S., Applegate, B., Bhunia, A., Waddell, J., and Singh, M. 2015. Thermal inactivation of non-O157 Shiga toxin producing Escherichia coli in laboratory media. IAFP annual meeting. Technical Talk (T11-06), Portland, OR.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Food safety researchers and scientistsin academia, industry and government Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Through publication of peer-reviewed articles and presentation in professional meetings What do you plan to do during the next reporting period to accomplish the goals? AIM 1. To optimize and improve sample preparation step prior to biosensor-based detection Optimizeparamagnetic beads based capture ofpathogens directly fromfood samplesor from enriched food samples prior to application to biosensor platforms. Also optimize sample enrichment steps, including, time, temperature and growth promoting agents AIM 2. Improve biosensor performance by employing novel probes, reagents and media Continue evaluation of various selective or differential media for Enterobacteriaceaeand other foodborne pathogens AIM 3. Optimize biosensor performance for pathogens Continue optimization of biosensors for detection of various pathogens from real world food samples

Impacts
What was accomplished under these goals? AIM 1. To optimize and improve sample preparation step prior to biosensor-based detection Antibodies were developed for use with paramagnetic beads for capture of Brucella directly from milk or from enriched food samples AIM 2. Improve biosensor performance by employing novel probes, reagents and media After evaluation of several selective agar media for use with laser light scattering sensor for specific pathogen detection and identification, we chose XLT4 for Salmonella, sorbitol MacConkey (SMAC) agar for Shiga toxin producing E. coli (STEC) serovars and phenol red mannitol (PRM) for Bacillus. AIM 3. Optimize biosensor performance for pathogens The light scattering sensor was able to identify and classify the colonies of top-20 serovars of Salmonella responsible for human infection and seven most important serovars of STEC strains growing on selective media in real-time. Serovar classification based on scatter pattern was in total agreement with genetic fingerprints. Light scattering sensor also successfully detected Salmonella from chicken rinse samples, STEC from lettuce and ground beef and Bacillus from raw milk in less than 24 h. In addition, Brucella spp. was successfully concentrated from milk using PMB and was detected by qPCR at 102 cfu/ml.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Singh, A.K., Bettasso, A.M., Bae, E., Rajwa, B., Dundar, M.M., Forstere, M.D., Liu, L., Barrette, B., Lovchike, J., Robinson, J.P., Hirleman, E.D. and Bhunia, A.K. 2014. Laser optical sensor, a label-free on-plate Salmonella enterica serovar colony-detection tool. mBio 5(1):e01019-13.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Niu, Y., Vemulapalli, R., and Bhunia, A.K. 2014. Immunological detection of Brucella species. International Association for Food Protection annual meeting, Indianapolis, IN, Aug 3-6, 2014; Abstr. P2-39
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Kim, H., Singh, A.K., Bhunia, A.K., and Bae, E., 2014. Laser-induced speckle scatter patterns in Bacillus colonies. Frontier in Microbiology. 5: 537 (1-9)
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Lathrop, A.A., Bailey, T.W., Kim, K.P., and Bhunia, A.K. 2014. Pathogen-specific antigen target for production of antibodies produced by comparative genomics, Antibody Technology Journal 4:4 (1-10)
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Bhunia, A.K. 2014. One day to one hour: how quickly can foodborne pathogens be detected? Future Microbiology (Invited), 9(8):935-946
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Cho, I-H., Radadia, A., Farrokhzad, K., Ximenes, E., Bae, E., Singh, A.K., Oliver, H., Ladisch, M.R., Bhunia, A.K., Applegate, B., Mauer, L., Bashir, R., Irudayaraj, J. 2014. Nano/Micro and spectroscopic approaches to food pathogen detection. Annu. Rev. Anal. Chem. 7:65-88
  • Type: Books Status: Published Year Published: 2014 Citation: Ray, B., and Bhunia, A.K. 2014. Fundamental Food Microbiology (Text book), 5th Edition, CRC Press (Taylor and Francis group), Boca Raton, FL
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Singh, A.K., Sarkar, P., Janaswamy, S., Yao, Y., and Bhunia, A.K. 2014. Encapsulation and Delivery of Antimicrobial Compounds, In Novel Food Preservation and Microbial Assessment Techniques, Chapter 8. Editor, Ioannis S. Boziaris, CRC Press, Boca Raton, FL. Pp. 218-236
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Drolia, R., Kim, H. and Bhunia, A.K. 2014. Listeria adhesion protein interaction with mammalian chaperone 60 promotes epithelial paracellular permeability through NF-kB activation. In American Society for Microbiology 114th General Meeting. May 17-21, 2014, Boston, MA: Abst # 2477
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Fleishman Littlejohn, A., Lim, T., Broady, A., Farrokhzad, K., Bhunia, A.K., Morgan, M., and Applegate, B. 2014. Efficacy of low level chlorine dioxide gas treatment on romaine and cantaloupe as indicated by microbial diversity. In American Society for Microbiology 114th General Meeting. May 17-21, 2014, Boston, MA: Abst # 2797
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lim, T., Broady, A., Jackson, J., Anderson, B., Fleishman Littlejohn, A., Jensen, J., Keener, K., Bhunia, A.K., and Applegate, B. 2014. Microbial diversity as an indicator of the efficacy of atmospheric cold plasma treatments of produce. In American Society for Microbiology 114th General Meeting. May 17-21, 2014, Boston, MA: Abst # 2798
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Singh, A., and Bhunia, A.K. 2014. Label-free optical biosensor to monitor antibiotic resistance in bacterial pathogens. International Association for Food Protection annual meeting, Indianapolis, IN, Aug 3-6, 2014; Technical Talk (T1-03)