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Yoo B.K.,Residue Chemistry and Predictive Microbiology Research Unit | Liu Y.,Molecular Characterization of Foodborne Pathogens Research Unit | Juneja V.,Residue Chemistry and Predictive Microbiology Research Unit | Huang L.,Residue Chemistry and Predictive Microbiology Research Unit | Hwang C.-A.,Residue Chemistry and Predictive Microbiology Research Unit
Food Control | Year: 2015

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen causing serious illnesses and hospitalizations in the United States. Bacteria that are exposed to environmental stresses during food processing may exhibit different growth patterns in the subsequent growth environment. The purpose of this study was to examine the effect of environmental stresses on the growth of O15H and non-O157 STEC in lettuce or cantaloupe. Strains of O157:H7 and non-O157 STEC (O26:H11, O103:H1, O104:H4, and O145:NM) were subjected to four selected stresses including 2ppm of chlorine, aw of 0.97 (osmotic stress), and pH 5(acid stress)at 22°C for 24h, or starvation (lack of nutrients) at 22°C for 15d. A cocktail mix of stressed or non-stressed (control) O157 or non-O157 at 3 log CFU/g (control or stressed) was inoculated on lettuce or cantaloupe and incubated at 10 and 22°C for four weeks. While there were significant differences (p<0.05) in the growth of stressed and unstressed cells of non-O157 STEC, no difference was observed in the growth of stressed and unstressed O157 STEC cells. Osmotic-stressed non-O157 STEC had significantly higher cell populations than control with 2 log difference (9.0 vs. 6.8 log CFU/g) at 10°C on lettuce and 1 log difference (9.3 vs. 8.3 log CFU/g) at 22°C on cantaloupe after 4 weeks. Acid-stressed non-O157 STEC had significantly higher cell populations than control at 10°C after 4 weeks with >1 log difference (7.7 vs. 6.3 log CFU/ml) on cantaloupe. Starvation-stressed non-O157 STEC showed significantly higher cell populations than control with 1 log difference (8.4 vs. 7.2 log CFU/g) at 22°C on cantaloupe after 4 weeks. The results indicated that osmotic, acid, or starvation stress may enhance the growth of non-O157 STEC on lettuce or cantaloupe and lead to a greater safety risk. © 2015.


Smith J.L.,Molecular Characterization of Foodborne Pathogens Research Unit | Liu Y.,Molecular Characterization of Foodborne Pathogens Research Unit | Paoli G.C.,Molecular Characterization of Foodborne Pathogens Research Unit
Canadian Journal of Microbiology | Year: 2013

Listeria monocytogenes, a major foodborne pathogen, possesses a number of mechanisms that enable it to combat the challenges posed by acidic environments, such as that of acidic foods and the gastrointestinal tract. One mechanism employed by L. monocytogenes for survival at low pH is the adaptive acid tolerance response (ATR) in which a short adaptive period at a nonlethal pH induces metabolic changes that allow the organism to survive a lethal pH. Overcoming acid conditions by L. monocytogenes involves a variety of regulatory responses, including the LisRK 2-component regulatory system, the SOS response, components of the σB regulon, changes in membrane fluidity, the F0F1-ATPase proton pump, and at least 2 enzymatic systems that regulate internal hydrogen ion concentration (glutamate decarboxylase and arginine deiminase). It is not clear if these mechanisms exert their protective effects separately or in concert, but it is probable that these mechanisms overlap. Studies using mutants indicate that the glutamate decarboxylase system can protect L. monocytogenes when the organism is present in acidic juices, yogurt, salad dressing, mayonnaise, and modified CO2 atmospheres. The glutamate decarboxylase system also has a role in protecting L. monocytogenes against the acidic environment of the stomach. There is a need to study other acid resistance mechanisms of L. monocytogenes to determine their effectiveness in protecting the organism in acidic foods or during transit through the acid stomach.


Suo B.,Henan Agricultural University | He Y.,Molecular Characterization of Foodborne Pathogens Research Unit | Irwin P.,Molecular Characterization of Foodborne Pathogens Research Unit | Gehring A.,Molecular Characterization of Foodborne Pathogens Research Unit
Food Analytical Methods | Year: 2013

DNA microarrays are promising high-throughput tools for multiple pathogen detection. Currently, the performance and cost of this platform has limited its broad application in identifying microbial contaminants in foods. In this study, an optimized custom DNA microarray with flexibility in design and content for foodborne pathogen detection was developed through the systematic evaluation of spotting buffers, probe lengths, scanning conditions, and nucleic acid amplification and labeling strategies. Briefly, by robotic contact printing, a spotting solution of 50 % dimethylsulfoxide produced uniform and high-quality spots on UltraGAPS glass slides coated with aminopropyl silane. The use of 60 % photomultiplier tube gain in scanning ∼70-mer oligonucleotide probes resulted in strong signals and low background. For sample preparation, multiplex PCR amplification coupled with fluorescent labeling of DNA using the Klenow fragment and random hexamers achieved higher specificity than whole genome random amplification. To minimize the cost of the assay, the quantities of probes, Klenow fragment, and Cy5 were substantially reduced in each assay without noticeably affecting the detection efficiency. Applying the optimized microarray assay to 26 fresh meat samples, three different isolates of Escherichia coli O157:H7 were found in four individual packages, demonstrating that the assay has a great potential for identifying and genotyping multiple pathogens in a real food system. © 2013 Springer Science+Business Media New York (outside the USA).


Gehring A.,Molecular Characterization of Foodborne Pathogens Research Unit
Sensors (Basel, Switzerland) | Year: 2013

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.


He Y.,Molecular Characterization of Foodborne Pathogens Research Unit | Ingudam S.,ICAR Research Complex for NEH Region | Reed S.,Molecular Characterization of Foodborne Pathogens Research Unit | Gehring A.,Molecular Characterization of Foodborne Pathogens Research Unit | And 2 more authors.
Journal of Nanobiotechnology | Year: 2016

Background: Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20nm) 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 1mg/ml, respectively. To completely inactivate 108-9 CFU/ml bacterial cells in 4h, a minimal concentration of 2mg/ml MgO nanoparticles was required for C. jejuni whereas E. coli O157:H7 and Salmonella Enteritidis required at least 8mg/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 (1.1μM) 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. © 2016 The Author(s).


Brewster J.D.,Molecular Characterization of Foodborne Pathogens Research Unit | Paul M.,U.S. Department of Agriculture
Journal of Dairy Science | Year: 2016

Centrifugation is widely used to isolate and concentrate bacteria from dairy products before assay. We found that more than 98% of common pathogenic bacteria added to pasteurized, homogenized, or pasteurized homogenized milk were recovered in the pellet after centrifugation, whereas less than 7% were recovered from raw milk. The remaining bacteria partitioned into the cream layer of raw milk within 5 min, and half-saturation of the cream layer required a bacterial load of approximately 5 × 108 cfu/mL. Known treatments (e.g., heat, enzymes or solvents) can disrupt cream layer binding and improve recovery from raw milk, but can also damage bacteria and compromise detection. We developed a simple, rapid agitation treatment that disrupted bacteria binding to the cream layer and provided more than 95% recovery without affecting bacteria viability. Combining this simple agitation treatment with a previously developed real-time quantitative PCR assay allowed the detection of Salmonella spp. in raw milk at 4 cfu/mL within 3 h. To our knowledge, this is the first report of an effective method for achieving high centrifugal recovery of bacteria from raw milk without impairing bacterial viability. © 2016 American Dairy Science Association.


PubMed | Zhengzhou University, Xuchang University and Molecular Characterization of Foodborne Pathogens Research Unit
Type: Comparative Study | Journal: Molecules (Basel, Switzerland) | Year: 2015

Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145 and Streptococcus agalactiae. False-positive results were observed in all 12 in-house real-time LAMP assays, while all the negative controls of Isothermal Master Mix remained negative after amplification. The detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg, whereas the sensitivity of the commercialized kit for E. coli O145 was 100 pg. In conclusion, the 12 in-house real-time LAMP assays were impractical to use, while the commercialized kit Isothermal Master Mix was useful for the detection of most bacterial pathogens.


PubMed | Molecular Characterization of Foodborne Pathogens Research Unit
Type: Journal Article | Journal: PloS one | Year: 2014

The biofilm life style helps bacteria resist oxidative stress, desiccation, antibiotic treatment, and starvation. Biofilm formation involves a complex regulatory gene network controlled by various environmental signals. It was previously shown that prophage insertions in mlrA and heterogeneous mutations in rpoS constituted major obstacles limiting biofilm formation and the expression of extracellular curli fibers in strains of Escherichia coli serotype O157:H7. The purpose of this study was to test strains from other important serotypes of Shiga toxin-producing E. coli (STEC) (O26, O45, O103, O111, O113, O121, and O145) for similar regulatory restrictions. In a small but diverse collection of biofilm-forming and non-forming strains, mlrA prophage insertions were identified in only 4 of the 19 strains (serotypes O103, O113, and O145). Only the STEC O103 and O113 strains could be complemented by a trans-copy of mlrA to restore curli production and Congo red (CR) dye affinity. RpoS mutations were found in 5 strains (4 serotypes), each with low CR affinity, and the defects were moderately restored by a wild-type copy of rpoS in 2 of the 3 strains attempted. Fourteen strains in this study showed no or weak biofilm formation, of which 9 could be explained by prophage insertions or rpoS mutations. However, each of the remaining five biofilm-deficient strains, as well as the two O145 strains that could not be complemented by mlrA, showed complete or nearly complete lack of motility. This study indicates that mlrA prophage insertions and rpoS mutations do limit biofilm and curli expression in the non-serotype O157:H7 STEC but prophage insertions may not be as common as in serotype O157:H7 strains. The results also suggest that lack of motility provides a third major factor limiting biofilm formation in the non-O157:H7 STEC. Understanding biofilm regulatory mechanisms will prove beneficial in reducing pathogen survival and enhancing food safety.


PubMed | Albany Research Center, NanoDetection Technology and Molecular Characterization of Foodborne Pathogens Research Unit
Type: Comparative Study | Journal: Toxins | Year: 2014

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.


PubMed | Molecular Characterization of Foodborne Pathogens Research Unit
Type: Journal Article | Journal: Sensors (Basel, Switzerland) | Year: 2015

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 10 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

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