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The USDA-FSIS has zero tolerance for E. coli O157:H7 in raw ground beef. Currently, FSIS collects samples from beef processing facilities and ships them overnight to regional testing laboratories. Pathogen detection requires robust methods that employ an initial 15–24 h culture enrichment. This study assessed the potential of using the ΦV10nluc phage-based luminescence detection assay during enrichment while the sample is in transit. Parameters including phage concentrations, temperature, and media-to-sample ratios were evaluated. Results in liquid media showed that 1.73× 103 pfu/mL of ΦV10nluc was able to detect 2 CFU in 10 h. The detection of E. coli O157:H7 was further evaluated in kinetic studies using ratios of 1:3, 1:2, and 1:1 ground beef sample to enrichment media, yielding positive results for as little as 2–3 CFU in 325 g ground beef in about 15 h at 37 °C. These results suggest that this approach is feasible, allowing the detection of a presumptive positive upon arrival of the sample to the testing lab. As the current cargo hold controlled temperature is required to be 15–25 °C, the need for elevated temperature should be easily addressed. If successful, this approach could be expanded to other pathogens and foods.

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.

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.

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.

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).

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.

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.

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.

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 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.

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.

In this manuscript we present a modified kinetic model for pathogen capture efficiency (E) variation with mixing time (Tmix) which is applicable for most combinations of immuno-magnetic bead (IMB)-target organism concentrations (e.g., [IMB] ˜ 8 × 106 IMB mL-1; Salmonella enterica serovar Enteritidis, [SE] ˜ 102-108 CFU mL-1). We found that as the ratio of [IMB] to [SE] was decreased from more than 105 to ca. 10-1, the average number of cells captured per IMB SE complex increased from ca. 1 to 4 ± 1. Concurrently, using a modified kinetic model for fitting E variation with Tmix, the maximum possible level of capture (§) was observed to decline from ˜ 100% (§˜ 1; [IMB] > > [SE]) to 23% (§˜ 0.23; [IMB]:[SE] ˜ 0.1). Using a purely kinetic approach we also found that an empirical mass transport term (yobs= (3.1 ± 1.2) × 10-9 mL IMB-1 min 1; averaged across all [SE] tested: [IMB]:[SE] ˜ 200000, 200, 3, and 0.1) did not differ much from yobs reported in earlier work (3.35 ± 0.2 × 10-9 mL min-1 IMB-1) at extremely low [SE] (e.g., ± 100 CFU mL-1). Upon inserting the classically-derived equation for γ into the kinetic model and solving for the effective IMB radius (rIMB), we found that the rIMB was 2.3 ± 0.16 μ (§ S; averaged across all [IMB]:[SE]) which was only 0.5–0.9 μ greater than the rIMB reported by the manufacturer (1.4 ± 0.2 μ). These results support previous work which showed a similar small rIMB deviation possibly due to the hydro-dynamic behavior of the IMBs in relatively dilute buffer suspensions ([IMB ˜ 8 × 106 IMB mL-1) and argues that IMB-based cell capture is almost exclusively a function of mass transport.

Background The disruption of the bacterial cell wall plays an important part in achieving quantitative extraction of DNA from Eubacteria essential for accurate analyses of genetic material recovered from environmental samples. Results In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70 × 106 (±9.05%), 4.77 × 108 (±31.0%), and 5.93 × 108 (±4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt; Gram-positive), Shigella sonnei (Ss; Gram-negative), and Escherichia coli O79 (Ec; Gram-negative). We have utilized real-time PCR (qPCR) quantification with two specific sets of primers associated with the 16S rRNA “gene” to determine the number of copies CFU-1 by comparing the unknown target DNA qPCR results with standards for each primer set. Based upon statistical analyses of our results, we determined that the Agencourt Genfind v2, High Pure PCR Template Prep Kit, and Omnilyse methods consistently provided the best yield of genomic DNA ranging from 141 to 934, 8 to 21, and 16 to 27 16S rDNA copies CFU-1 for Bt, Ss, and Ec. If one assumes 6–7 copies of the 16S rRNA gene per genome, between 1 and 3 genomes per actively dividing cell and ≥ 100 cells CFU-1 for Bt (found to be a reasonable assumption using an optical method expounded upon herein) or between 1 and 2 cells CFU-1 for either Ss or Ec, then the Omnilyse procedure provided nearly quantitative extraction of genomic DNA from these isolates (934 ± 19.9 copies CFU-1 for Bt; 20.8 ± 2.68 copies CFU-1 for Ss; 26.9 ± 3.39 copies CFU-1 for Ec). The Agencourt, High Pure, and Omnilyse technologies were subsequently assessed using 5 additional Gram-positive and 10 Gram-negative foodborne isolates (n = 3) using a set of “universal” 16S rDNA primers. Conclusion Overall, the most notable DNA extraction method was found to be the Omnilyse procedure which is a “bead blender” technology involving high frequency agitation in the presence of zirconium silicate beads. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0326-z) contains supplementary material, which is available to authorized users.

Campylobacter jejuni and Campylobacter coli are the two important species responsible for most of the Campylobacter infections in humans. Reliable isolation and detection of Campylobacter spp. from food samples are challenging due to the interferences from complex food substances and the fastidious growth requirements of this organism. In this study, a novel biosensor-based detection called BARDOT (BActerial Rapid Detection using Optical scattering Technology) was developed for high-throughput screening of Campylobacter colonies grown on an agar plate without disrupting the intact colonies. Image pattern characterization and principal component analysis (PCA) of 6909 bacterial colonies showed that the light scatter patterns of C. jejuni and C. coli were strikingly different from those of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. Examination of a mixed culture of these microorganisms revealed 85% (34/40) accuracy in differentiating Campylobacter from the other three major foodborne pathogens based on the similarity to the scatter patterns in an established library. The application of BARDOT in real food has been addressed through the analysis of Campylobacter spiked ground chicken and naturally contaminated fresh chicken pieces. Combined with real-time PCR verification, BARDOT was able to identify Campylobacter isolates from retail chicken. Moreover, applying passive filtration to food samples facilitated the isolation of pure Campylobacter colonies and therefore overcame the interference of the food matrix on BARDOT analysis.

A modified approach using a light addressable Potentiometric sensor (LAPS) method to detect Escherichia coli O157.H7 in solutions is described. The bacterial cells were captured by the specific antibody coated on magnetic beads. The immuno paramagnetic beads (IMB) were then concentrated by a magnetic separator. Fluorescein-labeled anti E. coli antibody and urease-conjugated anti-fluorescein antibody were then used to label the cells captured by the beads. After labeling, the mixtures were rapidly filtered through 0.45 or 1.0 μm nitrocellulose or polycarbonate filters. The production of NH3 from urea by the conjugated urease associated with the captured bacteria was then analyzed. The results indicate that IMB approach can be used to increase the specificity of the LAPS method for pathogenic bacteria detection. With this procedure, the presence of about one E. coli O157:H7 CFU per g of hamburger meat can be detected after a 6 h incubation.

Live cells of E. coli O157:H7 were treated with 4′,6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for observation by epifluorescent microscopy. The treated bacteria, which exhibited minimal growth activity and partially retained respiratory function, were captured by goat anti-E. coli O157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB). The beads with captured bacteria were then concentrated by magnetic separators. The efficiency of this magnetic concentration step was less than that of using high speed centrifugation. The antibody-captured and IMB-immobilized bacteria were then applied on HF-treated, bovine serum albumin (BSA)-coated microscope slides mounted on an automated stage, and magnetically aligned before fluorescence distribution was measured by a cooled CCD attached to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bacteria. This approach could reduce the enumeration time for and increase the efficiency of bacteria counting by manual efforts. The developed procedure of capturing, concentrating and enumerating E. coli O157:H7 exhibited a similar applicability in buffer, pork carcass wash solution and apple juice as described in this study. The presence of about 100 cells/mL of the bacteria could be detected in 30 min with developed procedure.

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.

For antibody generation, pathogenic bacteria are often heat-treated prior to inoculation into host animals in order to prevent infection and subsequently, premature death of the host. Inoculation of host rabbits with gamma radiationkilled pathogenic bacteria was employed with the hopes of generating antibodies that would have higher affinity, relative to antibodies raised against thermally denatured microorganisms, to live pathogens. The two antibody sets, raised against either heat-killed or irradiated bacterial cells, were compared for immunological response with live, heattreated, chemically-treated (i.e., bleached), and irradiated Escherichia coli O157:H7 and Salmonella bacteria. With the exception of the chemically-treated cell immunological response, both antibody sets yielded similar responses— low for irradiated cells, moderate for live cells, and high for heat-treated cells. The results suggested that thermal or chemical treatment of live pathogen containing food samples will elicit higher immunological responses with either antibody system, indicating potential application for detection of the presence of live bacteria in non-irradiated food systems. In addition, these findings also indicated that an immunoassay analysis of irradiated foods may lead to low signals that might be interpreted as indicating the presence of live cells (i.e., may lead to a false negative result).

Background We have evaluated the antimicrobial properties of Ag-based nanoparticles (Nps) using two solid phase bioassays and found that 10-20 μL of 0.3-3 μM keratin-stabilized Nps (depending on the starting bacterial concentration = CI) completely inhibited the growth of an equivalent volume of ca. 103 to 104 colony forming units per mL (CFU mL-1) Staphylococcus aureus, Salmonella Typhimurium, or Escherichia coli O157:H7 on solid surfaces. Even after one week at 37°C on solid media, no growth was observed. At lower Np concentrations (= [Np]s), visible colonies were observed but they eventually ceased growing. Results To further study the physiology of this growth inhibition, we repeated these experiments in liquid phase by observing microbial growth via optical density at 590 nm (OD) at 37°C in the presence of a [Np] = 0 to 10-6 M. To extract various growth parameters we fit all OD[t] data to a common sigmoidal function which provides measures of the beginning and final OD values, a first-order rate constant (k), as well as the time to calculated 1/2-maximal OD (tm) which is a function of CI, k, as well as the microbiological lag time (T). Performing such experiments using a 96-well microtitre plate reader, we found that growth always occurred in solution but tm varied between 7 (controls; CI = 8 × 103 CFU mL-1) and > 20 hrs using either the citrate-([Np] ~ 3 × 10-7 M) or keratin-based ([Np] ~ 10-6 M) Nps and observed that {∂tm/∂ [Np]}citrate ~ 5 × 107 and {∂tm/∂ [Np]}keratin ~ 107 hr·L mol-1. We also found that there was little effect of Nps on S. aureus growth rates which varied only between k = 1.0 and 1.2 hr-1 (1.1 ± 0.075 hr-1). To test the idea that the Nps were changing the initial concentration (CI) of bacteria (i.e., cell death), we performed probabilistic calculations assuming that the perturbations in tm were due to CI alone. We found that such large perturbations in tm could only come about at a CI where the probability of any growth at all was small. This result indicates that much of the Np-induced change in tm was due to a greatly increased T (e.g., from ca. 1 to 15-20 hrs). For the solid phase assays we hypothesize that the bacteria eventually became non-culturable since they were inhibited from undergoing further cell division (T > many days). Conclusion We propose that the difference between the solid and liquid system relates to the obvious difference in the exposure, or residence, time of the Nps with respect to the bacterial cell membrane inasmuch as when small, Np-inhibited colonies were selected and streaked on fresh (i.e., no Nps present) media, growth proceeded normally: e.g., a small, growth-inhibited colony resulted in a plateful of typical S. aureus colonies when streaked on fresh, solid media.

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 x 10(-4) ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.

A new fluorescent sandwich method for the detection of Escherichia coli O157:H7 was developed. Strepavidin coated magnetic beads and fluorescence beads reacted with biotinylated anti E. coli O157 antibodies to form the immuno magnetic beads (IMB) and immuno fluorescence beads (IFB), respectively. The E. coli bacteria captured by IMB were further labeled with IFB to form IMB M- ( E. coli O157:H7) N- IFB O sandwich complexes where the subscripts M, N and O were integral numbers. Using broth cultured E. coli O157:H7, the sandwich method was able to detect the bacteria at the level of ~ 10 3 to 10 4 CFU/mL. Known quantity of freshly cultured E. coli O157:H7 cells were added to ground beef obtained from local markets. The bacteria in inoculated beef patties were enriched in EC broth containing novobiocin. After enriched for 4 h at 40 °C, the developed IMB-IFB method was applied to detect the presence of E. coli O157:H7. The results demonstrated that the developed method could detect the presence of 1 CFU of E. coli O157:H7 per gram of ground beef.

Outbreaks of E. coli O157:H7 by the consumption of contaminated cantaloupes fruits have been documented. Pathogens harbored in the networked but porous veins in khaki colored skin are difficult to remove. Thus, sensitive and efficient methods are needed to detect the presence of E. coli O157:H7 in cantaloupes. In this work, known quantities of the E. coli were inoculated on cantaloupe skins or flesh at room temperature for 1 h. The contaminated samples were incubated in growth media at 37°C for 3.3h. The bacteria captured by magnetic beads coated with anti E. coli O157 antibodies were further sandwiched by second anti E. coli O157 antibodies containing peroxidase for chemiluminescent measurements of captured bacteria. Alternatively, the captured bacteria were treated with electron-shuttering reagent to detect the cellular level of NAD(P)H via bioluminescence. The detected enzyme activity (peroxidase) and the NAD(P)H were used to measure the presence of the pathogen. The results indicated both the chemiluminescence and the fluorescence methods, in 96 well microplate format, could be applied to detect the E. coli contamination of cantaloupes.

Commercially available alfalfa seeds were inoculated with low levels (~ 4 CFU/g) of pathogenic bacteria. The inoculated seeds were then allowed to sprout in sterile tap water at 22°C. After 48 hours, the irrigation water and sprouts were separately transferred to bovine heart infusion (BHI) media. The microbes in the BHI samples were allowed to grow for 4 hours at 37°C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E.coli O157 and/or Salmonella in the growth media. Separation and concentration of IMB-captured pathogens were achieved using magnetic separators. The captured E. coli O157:H7 and Salmonella spp were further tagged with europium (Eu) labeled anti-E. coli O157 antibodies and samarium (Sm) labeled anti-Salmonella antibodies, respectively. After washing, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent 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 Salmonella enterica from the seeds inoculated with ~ 4 CFU/g of the pathogens. Non-targeted bacteria, e.g., Aeromonas and Citrobacter exhibited essentially no cross reactivity. Since the pathogen detection from the sprouts was achieved within 6 hours, the developed methodology could be use as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella enterica in this popular salad food.

Testing of foods for the presence of pathogenic organisms requires methods different than those employed in the clinical laboratory. In foods there could be very low levels of pathogenic bacteria and the organisms are often in a poor physiological state because of exposure to stressful conditions in the food environment or to stresses encountered during food processing and storage. The organism must be identified in the presence of a large population of competing flora. Therefore, a period of enrichment culturing of the sample in liquid growth medium is usually required in order to allow the organism to recover from injury and to allow for selective growth of low numbers of the target bacteria to detectable levels. Traditional methods for detection and identification of pathogenic bacteria in foods and other samples have relied on the use of specific microbiological media to isolate and enumerate viable bacterial cells followed by a series of biochemical and serological tests for confirmation.