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Escherichia coli strain FEX669 was isolated from retail ground chicken and shown to contain the extraintestinal pathogenic E. coli (ExPEC) virulence genes sfaD/focC and iutA. Because this presumptive ExPEC strain was isolated from a retail food item and it was a weak biofilm former, it was characterized using whole-genome sequencing using the PacBio RS II platform., Escherichia coli strain FEX669 was isolated from retail ground chicken and shown to contain the extraintestinal pathogenic E. coli (ExPEC) virulence genes sfaD, focC, and iutA. Because this presumptive ExPEC strain was isolated from a retail food item and it was a weak biofilm former, it was characterized using whole-genome sequencing using the PacBio RS II platform. Genomic analysis showed that the FEX669 chromosome is 4,973,943 bp long, with a GC content of 50.47%, and is accompanied by a ColV plasmid that is 237,102 bp long, with a GC content of 50.49%.

Isolation of the pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis from foods typically rely on slow (10–21 day) “cold enrichment” protocols before confirmed results are obtained. We describe an approach that yields results in 39 h that combines an alternative enrichment method with culture on a non-selective medium, and subsequent identification of suspect colonies using elastic light scatter (ELS) analysis. A prototype database of ELS profiles from five Yersinia species and six other bacterial genera found in pork mince was established, and used to compare similar profiles of colonies obtained from enrichment cultures from pork mince samples seeded with representative strains of Y. enterocolitica and Y. pseudotuberculosis. The presumptive identification by ELS using computerised or visual analyses of 83/90 colonies in these experiments as the target species was confirmed by partial 16S rDNA sequencing. In addition to seeded cultures, our method recovered two naturally occurring Yersinia strains. Our results indicate that modified enrichment combined with ELS is a promising new approach for expedited detection of foodborne pathogenic yersiniae.

As the world population has grown, new demands on the production of foods have been met by increased efficiencies in production, from planting and harvesting to processing, packaging and distribution to retail locations. These efficiencies enable rapid intranational and global dissemination of foods, providing longer “face time” for products on retail shelves and allowing consumers to make healthy dietary choices year-round. However, our food production capabilities have outpaced the capacity of traditional detection methods to ensure our foods are safe. Traditional methods for culture-based detection and characterization of microorganisms are time-, labor- and, in some instances, space- and infrastructure-intensive, and are therefore not compatible with current (or future) production and processing realities. New and versatile detection methods requiring fewer overall resources (time, labor, space, equipment, cost, etc.) are needed to transform the throughput and safety dimensions of the food industry. Access to new, user-friendly, and point-of-care testing technologies may help expand the use and ease of testing, allowing stakeholders to leverage the data obtained to reduce their operating risk and health risks to the public. The papers in this Special Issue on “Advances in Foodborne Pathogen Analysis” address critical issues in rapid pathogen analysis, including preanalytical sample preparation, portable and field-capable test methods, the prevalence of antibiotic resistance in zoonotic pathogens and non-bacterial pathogens, such as viruses and protozoa.

The complete genome sequence data of S. aureus SJTUF_J27 isolated from seaweed in China is reported here. The size of the genome is 2.8 Mbp with 32.9% G + C content, consisting of 2614 coding sequences and 77 RNAs. A number of virulence factors, including antimicrobial resistance genes (fluoroquinolone, beta-lactams, fosfomycin, mupirocin, trimethoprim, and aminocoumarin) and the egc enterotoxin cluster, were found in the genome. In addition, the genes encoding metal-binding proteins and associated heavy metal resistance were identified. Phylogenetic data analysis, based upon genome-wide single nucleotide polymorphisms (SNPs), and comparative genomic evaluation with BLAST Ring Image Generator (BRIG) were performed for SJTUF_J27 and four S. aureus strains isolated from food. The completed genome data was deposited in NCBI׳s GenBank under the accession number CP019117, https://www.ncbi.nlm.nih.gov/nuccore/CP019117. Keywords: Staphylococcus aureus, Genome assembly, Whole genome sequencing (WGS), Virulence factor

Background Antibiotic resistance genes (ARGs) can spread among pathogens via horizontal gene transfer, resulting in imparities in their distribution even within the same species. Therefore, a pan-genome approach to analyzing resistomes is necessary for thoroughly characterizing patterns of ARGs distribution within particular pathogen populations. Software tools are readily available for either ARGs identification or pan-genome analysis, but few exist to combine the two functions. Results We developed Pan Resistome Analysis Pipeline (PRAP) for the rapid identification of antibiotic resistance genes from various formats of whole genome sequences based on the CARD or ResFinder databases. Detailed annotations were used to analyze pan-resistome features and characterize distributions of ARGs. The contribution of different alleles to antibiotic resistance was predicted by a random forest classifier. Results of analysis were presented in browsable files along with a variety of visualization options. We demonstrated the performance of PRAP by analyzing the genomes of 26 Salmonella enterica isolates from Shanghai, China. Conclusions PRAP was effective for identifying ARGs and visualizing pan-resistome features, therefore facilitating pan-genomic investigation of ARGs. This tool has the ability to further excavate potential relationships between antibiotic resistance genes and their phenotypic traits.

Determination of the microbial content in foods is important, not only for safe consumption, but also for food quality, value, and yield. A variety of molecular techniques are currently available for both identification and quantification of microbial content within samples, however, their success is often contingent upon proper sample preparation when the subject of investigation is a complex mixture of components such as foods. Because of the importance of sample preparation, the present study employs a systematic approach to compare the effects of four different separation techniques (glass wool, 50 &mu, m polypropylene filters, graphite felt, and continuous flow centrifugation (CFC)) on sample preparation. To define the physical effects associated with the use of these separation methods, a multifactorial analysis was performed where particle size and composition, both pre- and post- processing, were analyzed for four different food matrices including lean ground beef, ground pork, ground turkey and spinach. Retention of three important foodborne bacterial pathogens (Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes) was also examined to evaluate the feasibility of the aforementioned methods to be utilized within the context of foodborne pathogen detection. Data from the multifactorial analysis not only delineated the particle size ranges but also defined the unique compositional profiles and quantified the bacterial retention. The three filtration membranes allowed for the passage of bacteria with minimal loss while CFC concentrated the inoculated bacteria. In addition, the deposition and therefore concentration of food matrix observed with CFC was considerably higher for meat samples relative to spinach. However, filtration with glass wool prior to CFC helped clarify meat samples, which led to considerably lower amounts of solids in the CFC vessel post processing and an increase in the recovery of the bacteria. Overall, by laying a framework for the deductive selection of sample preparation techniques, the results of the study can be applied to a range of applications where it would be beneficial to scientifically guide the pairing of the criteria associated with a downstream detection method with the most advantageous sample preparation techniques for complex matrices such as foods.

A commercial “concentrating pipette” was scrutinized from the perspective of sample volume reduction to augment native bacterial quantitation and identification within food samples. Aspects related to the elution protocol were evaluated for their impact on bacteria recovery. The results demonstrated there was no significant effect from different diluents/eluents on the relative recovery rate. Further, results from a complementary technique, dynamic light scattering, imply that recoveries less than unity are likely due to an inability to completely elute the bacterial cells from the capillary filter as opposed to bacterial injury caused by sample processing. When concentrating PBS washes from thawed frozen vegetables and enumerating after culturing at either 30 or 37 °C, the recovery rates were only slightly lower than the simplified experimental conditions. However, in food wash samples, significantly more mass-normalized bacteria were recovered at the lower culture temperature. Nevertheless, the vegetable wash concentration process did not change the most probable isolate composition relative to that of the controls at either culture temperature. The study demonstrates the potential for the system to serve as an effective tool for bacterial recovery and concentration within water-based food systems such as equipment rinses, process wash water, and other samples coming from food processing plants.

Microfiltration of chicken extracts has the potential to significantly decrease the time required to detect Salmonella, as long as the extract can be efficiently filtered and the pathogenic microorganisms kept in a viable state during this process. We present conditions that enable microfiltration by adding endopeptidase from Bacillus amyloliquefaciens to chicken extracts or chicken rinse, prior to microfiltration with fluid flow on both retentate and permeate sides of 0.2 μm cutoff polysulfone and polyethersulfone hollow fiber membranes. After treatment with this protease, the distribution of micron, submicron, and nanometer particles in chicken extracts changes so that the size of the remaining particles corresponds to 0.4-1 μm. Together with alteration of dissolved proteins, this change helps to explain how membrane fouling might be minimized because the potential foulants are significantly smaller or larger than the membrane pore size. At the same time, we found that the presence of protein protects Salmonella from protease action, thus maintaining cell viability. Concentration and recovery of 1-10 CFU Salmonella/mL from 400 mL chicken rinse is possible in less than 4 h, with the microfiltration step requiring less than 25 min at fluxes of 0.028-0.32 mL/cm(2) min. The entire procedure-from sample processing to detection by polymerase chain reaction-is completed in 8 h.

DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the "Super-7" Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a sequence from a "Super-7" STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C, however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

Rapid methods have been developed as relatively faster alternatives to plate culture for the detection of pathogenic bacteria in foods. However, since most rapid methods are subject to logistical limitations (e.g., sample volume size, analysis time, matrix effects) and/or a detection scheme with insufficient sensitivity needed to detect very low levels of bacteria in foods, culture enrichment is often employed to increase the concentration of targeted pathogens prior to detection. Multiplexed rapid detection platforms, capable of simultaneous detection of different bacteria in a single sample, necessitate co-enrichment (or mixed culture enrichment) of as many different targeted microorganisms as possible in a timely manner. This investigation compares the growth of four major foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, and Yersinia enterocolitica) inoculated into pristine media or ground pork and enriched in various culture media. Initial results revealed that, after 24 h incubation, the growth of L. monocytogenes (the slowest-growing pathogen examined) was increased by approximately 1-log by the supplementation of Universal Preenrichment Broth with Casamino Acids and/or Oxyrase. Overnight (24 h) growth of L. monocytogenes in ground pork enrichment cultures was enhanced up to ca. 2-log by the addition of either Casamino Acids or Casamino Acids and Oxyrase for each of the tested growth media. Ultimately, an overnight culture of the inoculated pathogens in any of the selected media containing both Casamino Acids and Oxyrase was observed to yield target bacterial concentrations that were at sufficient levels (between 10e5 and 10e6 CFU/mL) for detection by most rapid methods.

An antibody microarray was developed to detect the “top six” non-O157 serogroups, O26, O45, O103, O111, O121, and O145 of Shiga toxin-producing Escherichia coli (STEC), that have been declared as adulterant in meat by the Food Safety and Inspection Service of the United States Department of Agriculture. The sensitivity of the array was 105 CFU and the limit of detection of each serogroup in artificially inoculated ground beef was 1–10 CFU following 12 h of enrichment. Optimal concentrations of antibodies for printing and labeling and bacterial dilutions for binding to the antibodies were assessed. The array utilized a minimal amount of antibodies and other reagents and may be utilized for screening of multiple target O groups of STEC in parallel, directly from enriched samples in less than 3 h. Furthermore, the antibody array provides the flexibility to include other O serogroups of E. coli and may be adopted for high throughput screening. The method is potentially applicable to detect the pathogenic STEC O groups of E. coli in meat and other food, thus improving food safety and public health.

Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the “Big Six” non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.

This chapter provides an overview regarding the advantages of virulotyping over historic serology-based, PCR, based on genes that identify an organism, or enzymatic and biochemical-based analyses of foodborne pathogens in clinical diagnostics and food industry microbiology testing. Traditional identification and characterization methods are designed to detect a given genus, species, serovar or genetic variant of bacteria via enzymatic, genetic or biochemical characterization. Virulotyping is a technique that identifies specific virulence genes within the pathogen’s genome; thus having the discriminatory power to differentiate between a virulent and an avirulent (nonpathogenic) strain of bacterium within a species. As an example, the United States Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) methods for detection, isolation and identification of Shiga toxin producing E. coli (STEC) use the presence of the eae and stx genes as an initial screen after selective culture enrichment to determine if an adulterant STEC may be present in a raw beef verification sample and as a confirmatory screen of STEC isolates. While virulence testing for STEC is just one example of potential advantages of virulotyping, further benefits may also be realized from virulotyping other common foodborne pathogens (e.g., Campylobacter spp., Salmonella enterica, and Listeria monocytogenes), should avirulent subgroups one day be identified. While PCR and microarray analyses are currently used to perform virulotyping, genome sequencing-based methods may be a more robust and discriminatory technique, when they become practicable for food safety testing and clinical diagnostics. A potential future virulotyping-based medical diagnostics and food testing system might be envisioned in the United States, which could prevent illnesses.

Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris. Once infected by the ΦV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo®). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76 × 102 pfu ml−1 are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23 × 103 pfu ml−1 of phage in selective culture enrichments of ground beef as a representative food matrix. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.

Background Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have considerable potential as antimicrobial agents in food safety applications due to their structure, surface properties, and stability. The aim of this work was to investigate the antibacterial effects and mechanism of action of MgO nanoparticles against several important foodborne pathogens. Results Resazurin (a redox sensitive dye) microplate assay was used for measuring growth inhibition of bacteria treated with MgO nanoparticles. The minimal inhibitory concentrations of MgO nanoparticles to 104 colony-forming unit/ml (CFU/ml) of Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella Enteritidis were determined to be 0.5, 1 and 1 mg/ml, respectively. To completely inactivate 108−9 CFU/ml bacterial cells in 4 h, a minimal concentration of 2 mg/ml MgO nanoparticles was required for C. jejuni whereas E. coli O157:H7 and Salmonella Enteritidis required at least 8 mg/ml nanoparticles. Scanning electron microscopy examination revealed clear morphological changes and membrane structural damage in the cells treated with MgO nanoparticles. A quantitative real-time PCR combined with ethidium monoazide pretreatment confirmed cell membrane permeability was increased after exposure to the nanoparticles. In a cell free assay, a low level (1.1 μM) of H2O2 was detected in the nanoparticle suspensions. Consistently, MgO nanoparticles greatly induced the gene expression of KatA, a sole catalase in C. jejuni for breaking down H2O2 to H2O and O2. Conclusions MgO nanoparticles have strong antibacterial activity against three important foodborne pathogens. The interaction of nanoparticles with bacterial cells causes cell membrane leakage, induces oxidative stress, and ultimately leads to cell death.

Rapid methods for testing foods for the presence of pathogenic bacteria typically suffer from poor sensitivity and therefore require large concentrations of the bacteria to be present for detection. Food contaminated with pathogenic bacteria may often contain only a very small number of the microorganisms making their direct detection very challenging even with existing state-of-the-art methods. Therefore prior to detection, it may be of pertinence to increase the number of potentially present pathogenic bacteria through growth in an appropriate culture medium. Furthermore, multiplexed testing for the presence of different bacteria in food samples necessitates the ability to simultaneously increase, through growth/culture, the concentration of each targeted bacterial pathogen to a detectable level. We have evaluated several commercially available and custom media preparations for their ability to support the simultaneous growth of the following bacteria: Escherichia coli O157:H7, Listeria monocytogenes , Salmonella enterica , and Yersinia enterocolitica . Growth conditions (primarily enrichment media formulation and incubation temperature) that resulted in multiplication of all four pathogens to ca. 1 × 10 5 cells/mL within 24 h or less were considered sufficient as a culture enrichment step prior to testing with most rapid methods. Axenic culture enrichment of all the bacteria for 18 h readily yielded concentrations significantly greater than 1 × 10 5 cells/mL for each of 5 different growth media. Mixed culture enrichment of the bacteria in pristine culture media and ground pork slurries indicated that several of the tested conditions appeared to be suitable for the growth of the selected bacteria to the targeted detection level, with the exception of L. monocytogenes in the ground meat (inoculated at 1.1 CFU/mL).

Incidental contamination of foods by pathogenic bacteria and/or their toxins is a serious threat to public health and the global economy. The presence of food-borne pathogens and toxins must be rapidly determined at various stages of food production, processing, and distribution. Producers, processors, regulators, retailers, and public health professionals need simple and cost-effective methods to detect different species or serotypes of bacteria and associated toxins in large numbers of food samples. This review addresses the desire to replace traditional microbiological plate culture with more timely and less cumbersome rapid, biosensor-based methods. Emphasis focuses on high-throughput, multiplexed techniques that allow for simultaneous testing of numerous samples, in rapid succession, for multiple food-borne analytes (primarily pathogenic bacteria and/or toxins).

Streptavidin-coated magnetic beads were conjugated with biotinylated capture antibodies to both Escherichia coli O157:H7 and Salmonella Typhimurium to form multi-pathogen capture immunomagnetic beads (IMB-M). The efficacy of these beads was investigated in both pure and mixed culture suspensions, as well as in inoculated spinach and ground beef. Using dual-label time-resolved fluorescence, a sandwich immunoassay consisting of europium- and samarium-labeled detection antibodies was used to measure the capture of E. coli and Salmonella, respectively. IMB-M was just as effective as the mixture of IMB against E. coli and Salmonella in capturing both organisms in pure culture and E. coli in mixed culture. Capture of Salmonella in mixed culture by IMB-M resulted in lower fluorescent signals, but comparable detection limit. In inoculated food samples, IMB-M was just as effective as the mixture of IMB specific for both target organisms in detecting 1 cfu/g of E. coli and 100 cfu/g of Salmonella after a 6-h enrichment at 37 °C. Multi-pathogen capture IMB have the potential to be convenient and important tools for multi-pathogen detection.

Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect &ge, 0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1&times, and 10&times, diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

The Micro Imaging Technology (MIT) 1000 Rapid Microbial Identification (RMID) System is a device that uses the principles of light scattering coupled with proprietary algorithms to identify bacteria after being cultured and placed in a vial of filtered water. This specific method is for pure culture identification of Listeria spp. A total of 81 microorganisms (55 isolates) were tested by the MIT 1000 System, of which 25 were Listeria spp. and 30 a variety of other bacterial species. In addition, a total of 406 tests over seven different ruggedness parameters were tested by the MIT 1000 System to determine its flexibility to the specifications stated in the MIT 1000 System User Guide in areas where they might be deviated by a user to shorten the test cycle. Overall, MIT concluded that the MIT 1000 System had an accuracy performance that should certify this Performance Test Method for the identification of Listeria spp. This report discusses the tests performed, results achieved, and conclusions, along with several reference documents to enable a higher understanding of the technology used by the MIT 1000 System.

Accompanied with the growth of Escherichia coli O157 : H7, there was a production of Shiga-like toxins by this pathogenic bacterium. Time-course studies indicated that the accumulation of toxins in the medium occurred mainly at the stationary phase of cell growth. The growth of E. coli O157 : H7 in culture media was not significantly affected by the presence of other bacteria, e.g., E. coli K-12, E. coli B6, Salmonella and Pseudomonas, even at high ratios. However, the production of Shiga-like toxins by E. coli O157 : H7 could be reduced by certain other bacteria, e.g., E. coli K-12, Pseudomonas aeruginosa but not Pseudomonas putida. These lowering effects by other background bacteria on the toxin production were also observed in experiments using regular and irradiated ground beef. The presence of analogs of quorum-sensing molecules such as homoserine lactone (HSL) and indole, in general decreased the production of toxins by E. coli O157 : H7. This decrease could be partially reversed by the presence of subinhibitory concentrations of antibiotics. The complex nature of the control of Shiga toxin production is discussed. PRACTICAL APPLICATIONS The toxic effects of pathogenic bacterium E. coli O157 : H7 are related to the production of Shiga-like toxins by the pathogen. Current studies on the influence of environmental factors such as cell density, presence of other bacteria, chemicals and food components indicated a complex influence on the production of Shiga-like toxins by E. coli O157 : H7. The observation that the presence of certain other bacteria could lower the ability of E. coli O157 : H7 to produce the toxins was of particular interest. The analyses of the results suggested several practical possibilities to lower the production of the toxins, and thus, the health risk of the pathogen to the general public.

The thixotropic-like properties of saline/glycerol drops, containing biotinylated capture antibodies, on streptavidin-coated glass slides have been investigated, along with their implications for bacterial detection in a fluorescent microarray immunoassay. The thixotropic-like nature of 60:40 saline-glycerol semisolid droplets (with differing amounts of antibodies) was observed when bacteria were captured, and their presence detected using a fluorescently-labeled antibody. Semisolid, gel-like drops of biotinylated capture antibody became liquefied and moved, and then returned to semisolid state, during the normal immunoassay procedures for bacterial capture and detection. Streaking patterns were observed that indicated thixotropic-like characteristics, and this appeared to have allowed excess biotinylated capture antibody to participate in bacterial capture and detection. When developing a microarray for bacterial detection, this must be considered for optimization. For example, with the appropriate concentration of antibody (in this study, 0.125 ng/nL), spots with increased diameter at the point of contact printing (and almost no streaking) were produced, resulting in a maximal signal. With capture antibody concentrations greater than 0.125 ng/nL, the excess biotinylated capture antibody (i.e., that which was residing in the three-dimensional, semisolid droplet space above the surface) was utilized to capture more bacteria. Similarly, when the immunoassay was performed within a hydrophobic barrier (i.e., without a coverslip), brighter spots with increased signal were observed. In addition, when higher concentrations of cells (∼10(8) cells/mL) were available for capture, the importance of unbound capture antibody in the semisolid droplets became apparent because washing off the excess, unbound biotinylated capture antibody before the immunoassay was performed reduced the signal intensity by nearly 50%. This reduction in signal was not observed with lower concentrations of cells (∼10(6) cells/mL). With increased volumes of capture antibody, abnormal spots were visualized, along with decreased signal intensity, after bacterial detection, indicating that the increased droplet volume detrimentally affected the immunoassay.

Immunomagnetic beads (IMB) were synthesized using anti-Escherichia coli O157 antibodies and magnetic beads of two different sizes (1 mm and 2.6 to 2.8 mm) that contained a streptavidin coating, activated carboxyl groups or tosylated surfaces. The synthesized IMB, together with a commercially available IMB, were used to capture different strains of E. coli O157:H7 and E. coli O157:NM. The E. coli capture was measured by the time resolved fluorescence (TRF) intensity using a sandwich assay which we have previously demonstrated of having a sensitivity of 1 CFU/g after 4.5 hour enrichment [1]. The analyses of measured TRF intensity and determined antibody surface concentration indicated that larger beads provided higher response signals than smaller beads and were more effective in capturing the target of interest in pure culture and ground beef. In addition, while each type of IMB showed different favorable capture of E. coli O157:H7, streptavidin-coated IMB elicited the highest response, on average. Streptavidin-coated IMB also provided an economic benefit, costing less than $0.50 per assay. The results could be used to guide the proper choice of IMB for applications in developing detection processes for E. coli O157:H7.

The types of chemical linkage used to bind antibodies to magnetic beads to form immunomagnetic beads (IMB) were compared in the capture and detection of Salmonella Enteriditis from egg white, egg yolk, and whole egg. Egg components were inoculated with outbreak strains of S. Enteriditis. After incubation under different conditions, IMBs derived from linking antibodies to core magnetic beads via biotin–streptavidin interactions, Schiff-base bonds and unspecified proprietary chemistry were used to capture S. Enteriditis. Europium-labeled anti-Salmonella antibodies completed the sandwich, and time-resolved fluorescence served as the means of detection. For the Salmonella isolated in stationary phase and cultured from universal pre-enrichment broth (UPB), the detection signal intensity was affected by the chemistry utilized to link the antibodies to IMB, with results varying among the three test strains. When S. Enteriditis was cultured in egg yolk alone, plating data were similar to those of the growth of S. Enteriditis in UPB. Egg white by itself did not support the growth of S. Enteriditis. The addition of UPB to egg white restored the growth of Salmonella and yielded stronger detection signals than from cultures obtained from UPB with egg yolk. The detection signals obtained from the immunoassay were less intense for cultures grown in egg yolk + UPB than from cultures grown in UPB alone. The lower detection signals elicited by all IMBs suggest the availability of the antigenic groups recognized by the antibodies on IMBs was reduced in the presence of egg yolk.

The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5'-isothiocyanate (5'-FITC) at pH 7.0 and 35 degrees C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5'-FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The "native" structure of aldolase was required for the selective modification by 5'-FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5'-FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5'-FITC modification of aldolase. In contrast, F-6-P, AMP, NADH, and NAD(+) as well as free lysine and most importantly, the 6'-isomer of FITC exhibited no competition with 5'-FITC for the labeling of aldolase. Alone, the 6'-isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5'-FITC-labeled and -unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5'-FITC-labeled a tryptic peptide corresponding to residues 112-134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5'-FITC.

Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 10(6) to 10(9) and ca. 10(7) to 10(9) cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.

Microarrays have been used for gene expression and protein interaction studies, but recently, multianalyte diagnostic assays have employed the microarray platform. We developed a microarray immunoassay for bacteria, with biotinylated capture antibodies on streptavidin slides. To complete the fluorescent sandwich immunoassay following capture of bacteria (Escherichia coli O157:H7), a fluorescein-labeled antibody was used to label antibody-bound bacteria. The assay time was less than 4 h. As this method was developed, it became apparent that several methodological factors markedly affected the results. Therefore, a series of experiments was conducted to investigate methodological factors that affected the assay, including analysis of variation in normal printing, use of a coverslip to contain sample exposure to the microarrayed antibodies, surface chemistries of capture antibody immobilization/attachment and assay reaction time. The use of a coverslip during immunological reactions reduced the fluorescent signal by approximately 50% compared with the use of an uncovered, hydrophobic barrier that was also used to contain sample solution during exposure to the microarray. Also, when protein G was used for capture antibody attachment, a lower signal was generated than when biotinylated capture antibody was used. At high bacterial concentrations (108 or 109 cells/mL), the assay could be shortened to less than 20 min. Antibody microarrays were effectively used to detect bacteria, but assay parameters markedly affected the results and required careful design.

A sandwich method was developed for the detection of Salmonella in shell egg contents. Different outbreak strains of Salmonella were used to inoculate shell egg contents at levels between 1 and 25 cfu/egg. Spiked eggs were then mixed with proper enrichment media before incubation at 37C for 4–20 h. After enrichment, the bacteria were first captured by the use of immunomagnetic beads (IMBs) coated with anti-Salmonella antibodies. The captured bacteria were further labeled with samarium (Sm)-conjugated anti-Salmonella antibodies. Sandwiched Salmonella were then treated with fluorescence enhancement solution that contained strong Sm chelators. The processes ranging from IMB capture to Sm chelation were performed using an automated KingFisher apparatus (Thermo Fisher Scientific, Inc., Waltham, MA). The fluorescence intensity of chelated Sm was then measured by time-resolved fluorescence (TRF). Using this approach, the presence of ∼1 cfu of outbreak strains of Salmonella Enteritidis per egg (∼50 g of egg content) could be detected after enrichment for 20 h at 37C. For higher levels of S. Enteritidis contamination, e.g., 10 cfu/50 g of egg content, the enrichment time could be reduced to 5 h at 37C. Thus, depending on desired sensitivity, the whole detection process could be completed within either an 8-h shift or 24 h. The results demonstrated that a combination of IMB capture and TRF measurement could be a rapid and sensitive method for S. Enteritidis detection in eggs.

Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

The pathogenic bacteria Escherichia coli O157:H7 has been associated with numerous cases of foodborne illnesses. Many rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. However, many of these methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. We developed a rapid method (termed enzyme-linked immunomagnetic chemiluminescence [ELIMCL]) that employed a relatively affordable and portable luminometer with a superparamagnetic microparticle-based enzyme-linked sandwich immunoassay. A key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format, thus allowing for potential confirmation of the pathogen. This work presented the development of ELIMCL that incorporated a one-step sandwich immunoassay that was demonstrated to have a limit of detection of approximately 1 e5–1 e6 live cells/mL of E. coli O157:H7 in pristine-buffered saline in approximately 1 h or at least 50 cells/mL of E. coli O157:H7 inoculated into ground beef and culture enriched for 5.5 h.

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.

A new fluorescent sandwich method for the detection of Escherichia coli O157:H7 in ground beef was developed. Immunomagnetic beads (IMBs) precoated with anti-E. coli O157:H7 antibody were used to capture and concentrate E. coli O157:H7 present in ground beef. Streptavidin-coated fluorescent beads with anti-E. coli O157:H7 to form immunofluorescent beads (IFBs) were used to detect the E. coli O157:H7 captured by the IMBs by forming IMBM–E. coli O157:H7N–IFBO sandwich complexes, where the subscripts M, N and O were variable but integral numbers. This sandwich technique was able to detect 500 cfu of the bacteria in a 0.5-mL sample taken from a quantified and diluted broth culture. The method was then applied to detect E. coli O157:H7 in artificially contaminated ground beef. Known quantities of freshly cultured E. coli O157:H7 cells were added to 25-g ground beef patties held in sterile bags with filters. The inoculated patties were stored at 7C overnight; 75 mL of E. coli broth was added to each sample, and the bags were incubated at 40C with shaking at 160 rpm for 4.5 h. After the enrichment, E. coli O157:H7 in the filtrate was captured and concentrated with IMBs and further conjugated with IFBs for detection. The results demonstrate that, after a 4.5-h enrichment period, the developed procedure can detect the presence of E. coli O157:H7 in ground beef previously spiked with 1 cfu/g.

Escherichia coli O157:H7 was inoculated into ground beef. Samples were incubated in E. coli broth plus novobiocin growth media at 37C for 5 h. Bacteria were captured by magnetic beads coated with anti-E. coli O157:H7 antibodies and were treated with menadione, a membrane-permeable electron-transfer shuttering reagent, to oxidize the cellular nicotinamide adenine dinucleotide phosphate (NAD[P]H). Alternatively, bead-captured bacteria were sandwiched by second anti-E. coli O157:H7 antibodies labeled with peroxidase. The level of NAD(P)H and the enzyme activity of antibody-bound peroxidase were then measured by luminol-linked luminescence. The treatment of the E. coli O157:H7 with a bacterial protein extraction reagent diminished the luminescence associated with cellular NAD(P)H but not the luminescence associated with the sandwiched peroxidase label. Thus, the NAD(P)H-linked luminescence, not the luminescence that was associated with the sandwiched complexes, was related to the viability of the E. coli O157:H7.

An immunomagnetic bead time-resolved fluorescence assay (IMB-TRF) was developed for the detection of Shiga-like toxins I and II (SLT I and II) from Shiga-like toxin-producing Escherichia coli (STEC). Twenty-five gram of ground beef patties were inoculated with 1 CFU/g of O157 or non-O157 (STEC) stored at 7C for 5 h and incubated with 100 mL added EC broth at 37C and 140 r.p.m. shaking for 20 h. Ten µg/mL of ofloxacin was added to the EC broth after 20 h of incubation to enhance toxin production. After 4 more hours of incubation, samples were removed and centrifuged for 10 min at 5000 r.p.m. The supernatant was removed and saved and a bacterial protein extraction reagent was used to lyse the cells in the pellet and release intracellular SLTs (Fraction I) which were then recombined with extracellular SLTs (Fraction II) present in the supernatant. Immunomagnetic beads coated with monoclonal antibodies specific to Shiga-like toxins I and II were applied to capture and concentrate the toxins and captured toxins were labeled with lanthanide-tagged antitoxin antibodies to from sandwiched complexes prior to detection by a TRF reader. TRF pathogen detection levels, defined as signals > or = 2x uninoculated samples, were achieved in all STEC inoculated samples and no cross-reactivity was observed with either nonpathogenic microflora present on the meat or nontoxin-producing E. coli K12 inoculated samples.

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli 0157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli 0157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 × 10 3 cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.

The cellular ATP content of fourteen freshly harvested bacteria including Bacillus, Campylobacter, Citrobacter, Escherichia, Lactobacillus, Listeria, Pediococcus, Pseudomonas, Salmonella, Streptococcus and Yersinia, was determined using a luciferin-Iuciferase bioluminescence approach. Incubation of bacteria with carbonyl cyanide meta-chlorophenyl hydrazone (CCCP), a membrane protonophore, prior to cell breakage substantially lowered the bioluminescence signals indicating a decrease of cellular ATP content. The addition of CCCP afrer cell breakage had no detectable effect on the ATP levels. This differential effect of CCCP was not observed using heat-killed bacteria, i.e., the ATP content was not affected by CCCP incubation. The CCCP egects on cellular ATP level were detectable in bacterial suspensions with concentrations ranging from 106 to Id CFU/mL. Upon cold storage, the ATP content, but not the population of viable bacteria, decreased. The ATP content could be paaially restored by the addition of glucose. The ATP content restored by the addition of glucose was also sensitive to CCCP treatment. These results demonstrated that viable bacterial cells can be differentiated from dead cells by their responses to membrane protonophores.

E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses. A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture media. In recent years, highly sensitive chemiluminescence has become a more affordable and portable detection method. Chemiluminescent detection has been coupled with the selectivity of antibodies, magnetic microparticle separation/isolation, and enzymatic signal amplification in order to develop a rapid method, termed enzyme-linked immunomagnetic chemiluminescence (ELIMCL). This work presents the application of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline with a detection limit of 7.6 x 10(3) for live cells in approx. 75 min assay time. The blocking agent casein and the surfactant Tween 20 were used to lower background luminescence and thus maximize signal-to-noise ratios. After 5.5 h of enrichment culture, ELIMCL was demonstrated to detect E. coli O157:H7 inoculated in ground beef at 10 CFU/g in a total assay time of about 7 h.

We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av DeltaG0=-20 +/- 4 kJ mol(-1)) even in the presence of casein [1% (w/v): DeltaG0=-18 +/- 3 kJ mol(-1); DeltaDeltaG0 approximately -2 kJ mol(-1)]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; DeltaG0=-19 +/- 2 kJ mol(-1)) was tested for nonspecific binding in the presence of iota-carrageenan (0.03-0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency xi (DeltaG0=-11 +/- 4 kJ mol(-1); DeltaDeltaG0 approximately -8 kJ mol(-1)). Other anionic polysaccharides (0.1% kappa-carrageenan and polygalacturonic acid) had no significant effect (av DeltaG0=-19 +/- 1 kJ mol(-1); DeltaDeltaG0 approximately 0 kJ mol(-1)). Varying iota-carrageenan from 0% to 0.02% resulted in xi significantly diminishing from 0.69 (e.g., 69% of the cells captured; DeltaG0=-19 +/- 3 kJ mol(-1)) to 0.05 (DeltaG0=-11 +/- 2 kJ mol(-1); DeltaDeltaG0 approximately -9 kJ mol(-1)) at about 0.03% iota-carrageenan where xi leveled off. An optimum blocking ability was achieved with 0.04% iota-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of iota-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.

Commercially available alfalfa seeds were inoculated with low levels (approximately 4 CFU/g) of pathogenic bacteria and sprouted at 25C. At 48 h, the spent irrigation water and sprouts were separately transferred to brain heart infusion (BHI) broth and enriched for 4 h at 37C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E. coli O157 or Salmonella in the enriched media. Separation and concentration of captured pathogens were achieved using magnetic particle concentrators (MPC). IMB captured E. coli O157:H7 and Salmonella spp. then formed sandwiched complexes with europium (Eu) labeled anti-E. coli O157 antibodies and samarium (Sm) labeled anti-Salmonella antibodies, respectively. After washing the complexes, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent Eu- and Sm-chelates. The specific time-resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and many Salmonella spp. from spent irrigation water or sprouts grown from contaminated seeds. Nontargeted bacteria, e.g., native microflora present on the untreated seeds and inoculated Aeromonas and Citrobacter, exhibited no cross-reactivity and counts were not significantly different from background fluorescence of the IMB alone. Since pathogen detection was achieved within 6 h, the assay could detect contamination levels as low as 4 CFU/g of seeds and it showed no cross-reactivity with nonpathogenic microflora present on the sprouts, the developed methodology could be used as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella spp. in sprouts and their irrigation water.