Pathogen enrichment device (PED) enables one-step growth, enrichment and separation of pathogen from food matrices for detection using bioanalytical platforms.

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, and 100 CFU/g of the 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 79-100% and was directly detected by lateral-flow immunoassay (LFIA), quantitative PCR (qPCR) and light scattering sensor with sample-to-result time of 8-24h with a detection limit of 1CFU/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.

[1]  B. Kuehn.  Salmonella cases traced to egg producers: findings trigger recall of more than 500 million eggs. , 2010, JAMA.

[2]  Mieke Uyttendaele,et al.  The challenge of merging food safety diagnostic needs with quantitative PCR platforms , 2011 .

[3]  B. Bisha,et al.  Flow‐through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a complex food system , 2009, Biotechnology journal.

[4]  A. Bhunia,et al.  Effect of environmental stresses on antibody‐based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes , 2006, Journal of applied microbiology.

[5]  Rashid Bashir,et al.  Targeted capture of pathogenic bacteria using a mammalian cell receptor coupled with dielectrophoresis on a biochip. , 2009, Analytical chemistry.

[6]  Lee-Ann Jaykus,et al.  Sample preparation: the forgotten beginning. , 2009, Journal of food protection.

[7]  Arun K. Bhunia,et al.  Fiber-Optic Biosensor Employing Alexa-Fluor Conjugated Antibody for Detection of Escherichia coli O157:H7 from Ground Beef in Four Hours , 2006, Sensors (Basel, Switzerland).

[8]  Biao Suo,et al.  Evaluation of a multiplex selective enrichment broth SEL for simultaneous detection of injured Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes , 2014, Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology].

[9]  M. Widdowson,et al.  Foodborne Illness Acquired in the United States—Major Pathogens , 2011, Emerging infectious diseases.

[10]  R. Smiley,et al.  The effects of competition from non-pathogenic foodborne bacteria during the selective enrichment of Listeria monocytogenes using buffered Listeria enrichment broth. , 2014, Food microbiology.

[11]  Euiwon Bae,et al.  On the sensitivity of forward scattering patterns from bacterial colonies to media composition , 2011, Journal of biophotonics.

[12]  T. Geng,et al.  Selective enrichment media affect the antibody-based detection of stress-exposed Listeria monocytogenes due to differential expression of antibody-reactive antigens identified by protein sequencing. , 2006, Journal of food protection.

[13]  R. Lindqvist.  Preparation of PCR samples from food by a rapid and simple centrifugation technique evaluated by detection of Escherichia coli O157:H7. , 1997, International journal of food microbiology.

[14]  J. Sninsky,et al.  Recent advances in the polymerase chain reaction , 1991, Science.

[15]  Wamiq Manzoor Ahmed,et al.  Classification of Bacterial Contamination Using Image Processing and Distributed Computing , 2013, IEEE Journal of Biomedical and Health Informatics.

[16]  F Allerberger,et al.  Update: Multinational listeriosis outbreak due to 'Quargel', a sour milk curd cheese, caused by two different L. monocytogenes serotype 1/2a strains, 2009-2010. , 2010, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[17]  M. Erickson.  Microbial Risks Associated with Cabbage, Carrots, Celery, Onions, and Deli Salads Made with These Produce Items. , 2010, Comprehensive reviews in food science and food safety.

[18]  I G Wilson,et al.  Inhibition and facilitation of nucleic acid amplification , 1997, Applied and environmental microbiology.

[19]  Shu-I Tu,et al.  Evanescent Wave Fiber Optic Biosensor for Salmonella Detection in Food , 2009, Sensors.

[20]  J. Paul Robinson,et al.  Laser Optical Sensor, a Label-Free On-Plate Salmonella enterica Colony Detection Tool , 2014, mBio.

[21]  Y. Etoh,et al.  Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis , 2010, International journal of microbiology.

[22]  V. Hill,et al.  Comparison of hollow-fiber ultrafiltration to the USEPA VIRADEL technique and USEPA method 1623. , 2009, Journal of environmental quality.

[23]  A. Bhunia,et al.  - Nano-Optical Sensors for Food Safety and Security , 2016 .

[24]  R. G. Kroll,et al.  The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods. , 1992, The Journal of applied bacteriology.

[25]  T. Murakami.  Filter-based pathogen enrichment technology for detection of multiple viable foodborne pathogens in 1 day. , 2012, Journal of food protection.

[26]  Xiaojun Feng,et al.  Agarose-based microfluidic device for point-of-care concentration and detection of pathogen. , 2014, Analytical chemistry.

[27]  Sarah M. Schlemmer,et al.  Laboratory and pilot-scale dead-end ultrafiltration concentration of sanitizer-free and chlorinated lettuce wash water for improved detection of Escherichia coli O157:H7. , 2014, Journal of food protection.

[28]  J. Hoorfar,et al.  Automated 5′ Nuclease PCR Assay for Identification of Salmonella enterica , 2000, Journal of Clinical Microbiology.

[29]  Euiwon Bae,et al.  Label-free, non-invasive light scattering sensor for rapid screening of Bacillus colonies. , 2015, Journal of microbiological methods.

[30]  R. Scharff,et al.  Economic burden from health losses due to foodborne illness in the United States. , 2012, Journal of food protection.

[31]  Avraham Rasooly,et al.  Detection of campylobacter and Shigella species in food samples using an array biosensor. , 2004, Analytical chemistry.

[32]  L. Gould,et al.  Outbreaks associated with cantaloupe, watermelon, and honeydew in the United States, 1973-2011. , 2014, Foodborne pathogens and disease.

[33]  M. Josefsen,et al.  Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples. , 2004, Journal of microbiological methods.

[34]  S. Backert,et al.  A simplified and cost-effective enrichment protocol for the isolation of Campylobacter spp. from retail broiler meat without microaerobic incubation , 2011, BMC Microbiology.

[35]  Arun K. Bhunia,et al.  SEL, a Selective Enrichment Broth for Simultaneous Growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes , 2008, Applied and Environmental Microbiology.

[36]  Lee-Ann Jaykus,et al.  Bacterial Separation and Concentration from Complex Sample Matrices: A Review , 2004, Critical reviews in microbiology.

[37]  Michael R. Ladisch,et al.  Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration , 2013, Applied and Environmental Microbiology.

[38]  M. Wagner,et al.  Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay. , 2001, Research in microbiology.

[39]  Harsh Sharma,et al.  Review of biosensors for foodborne pathogens and toxins , 2013 .

[40]  P. Tomasula,et al.  Removal of Salmonella Enteritidis from commercial unpasteurized liquid egg white using pilot scale cross flow tangential microfiltration. , 2010, International journal of food microbiology.

[41]  Bartek Rajwa,et al.  Light Scattering Sensor for Direct Identification of Colonies of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145 and O157 , 2014, PloS one.

[42]  F. Tenover,et al.  A 5′ Nuclease PCR (TaqMan) High-Throughput Assay for Detection of the mecA Gene in Staphylococci , 2000, Journal of Clinical Microbiology.

[43]  Il-Hoon Cho,et al.  Membrane filter-assisted surface enhanced Raman spectroscopy for the rapid detection of E. coli O157:H7 in ground beef. , 2015, Biosensors & bioelectronics.

[44]  R. Holley,et al.  Factors influencing the microbial safety of fresh produce: a review. , 2012, Food microbiology.

[45]  R. Levin,et al.  Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment. , 2011, Food microbiology.

[46]  T. Popović,et al.  Development of a Real-Time Fluorescence PCR Assay for Rapid Detection of the Diphtheria Toxin Gene , 2002, Journal of Clinical Microbiology.

[47]  R. Knutsson,et al.  Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment , 2004, Applied and Environmental Microbiology.

[48]  Michael R Ladisch,et al.  Mechanistic study of membrane concentration and recovery of Listeria monocytogenes. , 2005, Biotechnology and bioengineering.

[49]  A. Bhunia,et al.  Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths , 2008, Journal of applied microbiology.

[50]  J. Paul Robinson,et al.  Light‐scattering sensor for real‐time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate , 2012, Microbial biotechnology.

[51]  P. Banerjee,et al.  Development of a rapid capture-cum-detection method for Escherichia coli O157 from apple juice comprising nano-immunomagnetic separation in tandem with surface enhanced Raman scattering. , 2014, International journal of food microbiology.

[52]  S Morabito,et al.  Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. , 2011, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[53]  J. Paul Robinson,et al.  Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species. , 2007, Biosensors & bioelectronics.

[54]  Yanbin Li,et al.  Interdigitated array microelectrode based impedance biosensor coupled with magnetic nanoparticle-antibody conjugates for detection of Escherichia coli O157:H7 in food samples. , 2007, Biosensors & bioelectronics.

[55]  Khalil Arshak,et al.  An overview of foodborne pathogen detection: in the perspective of biosensors. , 2010, Biotechnology advances.

[56]  D. Maki.  Coming to grips with foodborne infection--peanut butter, peppers, and nationwide salmonella outbreaks. , 2009, The New England journal of medicine.

[57]  S. Hossain,et al.  Multiplexed paper test strip for quantitative bacterial detection , 2012, Analytical and Bioanalytical Chemistry.

[58]  L. Jaykus,et al.  Detection of pathogens in foods: the current state-of-the-art and future directions , 2011, Critical reviews in microbiology.

[59]  Arun K Bhunia,et al.  One day to one hour: how quickly can foodborne pathogens be detected? , 2014, Future microbiology.

[60]  M. Griffiths,et al.  Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR , 2006, Applied and Environmental Microbiology.

[61]  Andrew Gehring,et al.  Rapid identification and classification of Campylobacter spp. using laser optical scattering technology. , 2015, Food microbiology.

[62]  T. Livache,et al.  Simultaneous enrichment and optical detection of low levels of stressed Escherichia coli O157:H7 in food matrices , 2014, Journal of applied microbiology.

[63]  A. Gehring,et al.  Mixed culture enrichment of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica , 2012 .

[64]  Barbara E. Mahon,et al.  Multistate Outbreak of Listeriosis Associated with Jensen Farms Cantaloupe—United States, August–September 2011 , 2011, MMWR. Morbidity and mortality weekly report.

[65]  J. Paul Robinson,et al.  Label-free detection of multiple bacterial pathogens using light-scattering sensor. , 2009, Biosensors & bioelectronics.

[66]  Arun K Bhunia,et al.  Biosensors and bio-based methods for the separation and detection of foodborne pathogens. , 2008, Advances in food and nutrition research.

[67]  Il-Hoon Cho,et al.  Rapid pathogen detection by lateral-flow immunochromatographic assay with gold nanoparticle-assisted enzyme signal amplification. , 2015, International journal of food microbiology.