Progress 04/01/11 to 03/31/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a na�ve library of biorecognition elements for bacterial typing-An alternative to �molecular typing.� Approach (from AD-416): This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/ or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations. Progress was made on the four objectives of this project (which falls under National Program 108) by ARS researchers at Wyndmoor, Pennsylvania with the goal to develop accurate, multiplexed methods for the detection and identification of pathogenic, foodborne bacteria. For Objective 1, ARS researchers in Wyndmoor, Pennsylvania developed methods for separating and concentrating pathogenic bacteria from various food matrices (e.g., using leukocyte removal filter, capacity of >100 mL 10% ground beef slurry, flow rate of > 10 mL/min, and < 5% retention of E. coli O157:H7; approximately 1:1,000 V/V concentration). Foods, and harmless bacteria in foods can interfere with accurate detection of harmful pathogens. Rapid filtration and centrifugation methods were developed that not only separate, but also concentrate target pathogens (E. coli, Salmonella, Listeria monocytogenes, and Campylobacter spp.) from various food matrices including ground poultry and beef. Food regulators and producers will benefit immensely from applying these techniques since testing for harmful pathogens would be streamlined in terms of expense and time. The ARS researchers primarily responsible for Objective 2 and Subobjective 4A retired before the project's completion. Nonetheless, progress was made in ensuring the ability to detect harmful Yersinia pestis in food. Accurate detection of virulent Y. pestis requires Y. pestis to have an intact virulence plasmid (small, circular strand of DNA) . However, Y. pestis may lose this plasmid if it is stressed during growth enrichment and/or isolation. Methods were developed to study the stability of the plasmid during growth of Y. pestis in raw ground meats (pork and beef). The toxicity of Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis bacteria depends on the presence of a small, circular piece of DNA inside the cell, which is referred to as a virulence plasmid (pYV). The pYV is unstable in all three bacteria and if it lost during growth or processing, the bacteria may not be properly detected or identified. In order to better understand pYV stability, a procedure to monitor its presence in Yersinia bacteria was developed. The procedure exploits the low calcium response (Lcr) and Congo red (CR) binding by the bacteria. The Lcr-CR positive bacteria (pYV-bearing strains) were used to study the growth of and to monitor the pYV stability of virulent Y. pestis and Y. pseudotuberculosis in raw ground beef. For Objective 3, further collaboration with a company that involved using monoclonal antibodies generated by our colleagues at USDA-Western Regional Research Center (WRRC) in Albany, California, resulted in the characterization and validation of anti-Shiga toxin 1 and 2 (Stx 1 and 2) antibodies using a commercial ELISA (enzyme-linked immunosorbent assay) platform. The antibodies were demonstrated to successfully interact with multiple subtypes of Stx 1 and 2 as well as detect STECs in incurred food and environmental samples. A novel biosensor-based method utilizing the BARDOT system was generated for high-throughput detection of Campylobacter spp. colonies. A laser-scattering image library was created by scanning over 7000 bacterial colonies. Based on the image library, BARDOT was able to automatically scan and classify a large number of Campylobacter spp. colonies, and differentiate them from other major foodborne pathogens including Salmonella, E. coli O157:H7, and L. monocytogenes. Considerable success was achieved in applying BARDOT to real food samples by means of passive filtration to overcome the interferences from the food matrix. Campylobacter jejuni and C. coli were rapidly detected and quantified in mixed cultures and spiked/ naturally contaminated chicken samples using a most probable number technique in combination with a multiplex qPCR assay. High detection sensitivity (8 cells/500g sample) along with a low error rate (13%) was demonstrated. A multiplex qPCR detection method for Salmonella spp., E. coli O157, and L. monocytogenes was developed and successfully validated by FDA scientists with a variety of FDA-regulated foods including cheeses, fresh fruits, and vegetables. Finally, an identification method for foodborne pathogens using BARDOT in combination with multiplex qPCR for the prescreening and identification of pathogens (Campylobacter jejuni and C. coli) in foods was developed. For Objective 4, in conjunction with collaborators at Shanghai Jiao Tong University (China), three genetic classification or typing schemes were compared for their ability to distinguish between pathogenic strains of S. aureus. Staphylococcus aureus isolates (N=108) were characterized and genotyped using Pulsed-Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST), and enterotoxin gene typing techniques. The results from all three typing methods consistently showed high genetic diversity of these strains from various geographic locations. This study also found high prevalence of staphylococcal enterotoxin (SE) genes and variability of the 18 SE gene types in these isolates, suggesting a potential use of SE genotyping method for discriminatory typing of S. aureus strains and tracking of foodborne outbreaks. Two novel, high- throughput antibody microarray techniques were developed: 1) arrayed capture antibodies and a universal bacterial label with traditional fluorescence scanning; and 2) a colorimetric sandwich ELISA comprised of arrayed capture antibodies, sample target (e.g., Shiga toxin) �sandwiched� between the capture and enzyme-associated reporter antibodies, and very inexpensive office flatbed page scanning. The two rapid, approximately 2 h, antibody-based microarray typing methods have been developed and applied to the simultaneous serotyping of various STEC and toxin typing of STECs induced to generate and release toxin during growth in the presence of low levels of a stress-inducing antibiotic. The former system has already found application for evaluation of typing antibodies produced by Kirkegaard Perry Laboratories (Gaithersburg, Maryland), Abraxis (Warrington, Pennsylvania), and Pennsylvania State University�s E. coli Reference Center (State College, Pennsylvania). The latter technique may find wide use in the field in large part due to its simplicity, high-throughput format, and low cost. Antibody and DNA typing/identification microarray platforms were developed using both glass and relatively inexpensive polystyrene plastic substrates. The systems, using either antibodies or computer-designed nucleic acid probes/ primers, interrogate large numbers of samples for E. coli O157:H7 and/or the �Big Six� non-O157 STECs. Total assay times were typically under 3 h with real-time (non-growth enriched samples) with relatively low detection limit. Finally, next-generation sequencing platforms to further the genotyping and characterization of Campylobacter from food were utilized. Dozens of Campylobacter strains were isolated from retail chickens and beef livers by using selective enrichment and a passive filtration technique. Whole genome sequencing was performed using Miseq and Ion Torrent sequencers. In addition, Campylobacter large-fragment libraries were constructed and Sanger sequenced in order to fill sequence gaps and prevent misassembly for the completion of Campylobacter genomes. Through bioinformatics analysis, a genome-level phylogenetic tree was obtained, which revealed that the genetic divergence of these strains was correlated with their food sources. The completed and annotated genome sequence of C. jejuni YH001 has been deposited at the NCBI GenBank sequence database under the accession no. CP015528 and made publicly available. For Objective 4A, efforts are continuing on determining if the portable Oxford Nanopore MinIon sequencer can be used to rapidly detect pathogenic bacteria in food samples. Currently, standardization efforts have been successful with control sequences. In addition, the MinIon has already found application for genome sequence closure for previously mentioned Campylobacter spp. Accomplishments 01 Rapid and effective means to separate and concentrate pathogens from food for subsequent detection. Pathogens may be found in food at low concentrations, and thus it is important to use techniques that are able to recover the pathogens so that they can be detected. ARS researchers in Wyndmoor, Pennsylvania developed methods, including the use of a leukocyte removal filter for separating and concentrating pathogenic bacteria from various food matrices. Also, food components and harmless bacteria in foods can interfere with accurate detection of harmful pathogens. Rapid filtration and centrifugation methods that not only separate, but also concentrate target pathogens (E. coli, Salmonella, Listeria monocytogenes, and Campylobacter spp.) from various food matrices including ground poultry and beef were developed. Food regulators and producers will benefit from applying these techniques since testing for harmful pathogens would be streamlined in terms of expense and time. 02 Effect of environmental factors and growth conditions on ability to detect Yersinia. Environmental factors can alter the characteristics of certain foodborne pathogens and impact the ability to detect them. Accurate detection of disease-causing bacteria belonging to the genus Yersina requires the pathogens to have an intact virulence plasmid (small, circular strand of DNA) known as pYV. However, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis may lose this plasmid under certain growth conditions, and the bacteria may not be accurately detected or identified. ARS researchers in Wyndmoor, Pennsylvania developed methods to study the stability of the plasmid and monitor its presence during growth of the pathogens in raw ground meats (pork and beef), providing a means to prevent the loss of the plasmid during growth conditions. The procedure exploits the low calcium response and Congo red binding properties of Yersinia. This work greatly enhances the ability to detect disease-causing Yersinia species in food, and this information will assist producers and regulators in reducing the incidence of Yersinia in foods. 03 A high-throughput method to detect Shiga toxins produced by Shiga toxin- producing E. coli (STEC). STEC produce very dangerous toxins, known as Shiga toxins, and methods to detect these toxins, including all of the genetic variants are needed for food and clinical testing. ARS researchers in Wyndmoor, Pennsylvania developed a novel, rapid detection method for Shiga toxins 1 and 2 (Stx1 and Stx2) using a high- throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following reaction of HRP with a precipitating chromogenic substrate, formation of colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric microarray method was very rapid and sensitive. The method could also be used to detect Shiga toxins in food, including ground beef. This method is very useful for the food and medical industries to detect the presence of STEC producing very dangerous types of Shiga toxins, thus improving food safety and the ability to diagnose infections. 04 Characterization of Campylobacter isolates from retail beef and poultry packages through DNA sequencing. Campylobacter is a foodborne pathogen and one of the most common causes of human gastrointestinal disease; it is prevalent in poultry, as well as other meat products. ARS researchers at Wyndmoor, Pennsylvania recovered a total of 27 new Campylobacter strains from various packages of chicken and beef liver. The genomes (the complete set of DNA of an organism) of all the Campylobacter strains were sequenced using next-generation sequencing technologies and assembled. Whole genome sequences and protein annotations of a C. jejuni and C. coli strains were completed and deposited into the NCBI Genbank database. Whole genome level comparison showed a significant genetic difference between C. jejuni and C. coli species, and isolates from the same food source appeared to be related more closely to each other than those from different sources. The finding of key virulence genes and multidrug resistant genes suggests the potential disease-causing potential and drug resistance of the strains. The whole genome sequences and annotations obtained in this study enhance the understanding of the genetic basis of Campylobacter pathogenesis and drug resistance, and also provides information on targets for design of more effective control strategies. 05 Use of magnesium oxide nanoparticles to inactive foodborne pathogens. There is a need for more effective strategies to control foodborne pathogens. ARS researchers in Wyndmoor, Pennsylvania investigated and revealed a strong antimicrobial activity on the foodborne pathogens, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella, with the use of magnesium oxide (MgO) nanoparticles. Studies of the antimicrobial mechanism of MgO nanoparticles showed the production of hydrogen peroxide in the nanoparticle suspension, as well as nanoparticle-induced oxidative stress in the bacterial cells. The strong antimicrobial effect of MgO nanoparticles implies that they have a potential use in food systems to control these and other pathogens. The understanding of the molecular basis of MgO nanoparticle action in bacteria could lead to the development of more powerful but less toxic antimicrobial nanoparticles for food safety intervention applications.
Impacts
(N/A)
Publications
- He, Y., Yan, X., Reed, S.A., Xie, Y., Chen, C., Irwin, P.L. 2015. Complete genome sequence of Campylobacter jejuni YH001 from beef liver which contains a novel plasmid. Genome Announcements. 3(1):e01492-14.
- Bhaduri, S., Chaney, K.J., Smith, J.L. 2011. A procedure for maintenance of the virulence plasmid (pYV) in Yersinia pestis under culture conditions. Foodborne Pathogens and Disease. 8:459-463.
- Wang, D., Brewster, J.D., Paul, M., Tomasula, P.M. 2015. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules. 20:4048-6059. DOI: 10.3390/molecules20046048.
- He, Y., Ingudam, S., Reed, S.A., Gehring, A.G., Strobaugh Jr, T.P., Irwin, P.L. 2016. Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens. Journal of Nanobiotechnology (Biomed Central Open Access). DOI:10.1186/s12951-016- 0202-0.
- Irwin, P.L., Nguyen, L.T., Chen, C., He, Y. 2014. Variability in DNA polymerase efficiency: effects of random error, DNA extraction method, and isolate type. JSM Mathematics & Statistics. 1(1):1003.
- Irwin, P.L., Nguyen, L.T., He, Y., Paoli, G., Gehring, A.G., Chen, C. 2014. Near-quantitative extraction of genomic DNA from various food-borne eubacteria. BMC Microbiology. DOI: 10.1186/s12866-014-0326-z.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a na�ve library of biorecognition elements for bacterial typing-An alternative to �molecular typing.� Approach (from AD-416): This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/ or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations. Progress was made on three of the four objectives of this project (falls under National Program 108) by ARS researchers at Wyndmoor, Pennsylvania, with the goal to develop accurate methods for the detection and identification (ID) of pathogenic, foodborne bacteria. Successful detection/ID typically necessitates concentration via physical means (e.g. , filtration) or increasing the number of factors (or biomarkers) through non-deleterious cell growth or genetic material amplification (i.e., PCR). For Objectives 1A and 1B, research has focused on the separation and concentration of Salmonella from ground poultry using filtration with leukocyte reduction filters. No additional research related to Objective 2 was conducted during the relevant time frame. For Objective 3, studies have progressed on the generation of high-throughput, multiplexed immunoassays for foodborne pathogens. Specifically, collaborative efforts using ELISA-based platforms produced by Abraxis (Warrington, Pennsylvania) using monoclonal antibodies generated by colleagues at USDA Western Regional Research Center (Albany, California). For Objective 4, the next-generation sequencing technique was used for rapid detection, genotyping, and characterization of Campylobacter from food. Dozens of Campylobacter strains were isolated from retailed chickens and beef livers by using selective enrichment and passive filtration technique. Whole genome sequencing was performed using Ion Torrent PMG sequencer. In addition, Campylobacter large-fragments libraries were constructed and Sanger sequenced in order to fill sequence gaps and prevent mis-assembly. Through bioinformatics analysis, we obtained the genome-level phylogenetic tree and revealed that the genetic divergence of these strains was correlated with their food sources. A novel plasmid encoding Type III secretion system, an important virulence factor, was found in the C. jejuni YH001strain. We also identified 10 unique lipid A components of lipopolysaccharides (LPSs, endotoxin) in these strains via functional genomic comparison. Combined with the sequence information in public databases, we found over 37 different types of C. jejuni/C. coli LPS antigens, indicating a high genetic variability in the genes contributing to pathogen virulence. The completed and annotated genome sequence of C. jejuni YH001 has been deposited at the NCBI GenBank sequence database under the accession no. CP010058 and made publicly available. A manuscript summarizing this work has been published recently. For Objective 4A, we are also assessing whether the Oxford Nanopore MinIon sequencer can be used to detect pathogenic bacteria in food samples. In addition for Objective 4B, since previous efforts were unsuccessful, we have developed and have been using a modified cell-SELEX protocol to isolate DNA aptamers that are specific for the E. coli serotype O157:H7 and each of the �Big Six� non-O157 Shiga toxin-producing serotypes. Currently we are at the 10th round of selection and have started sequencing aptamer pools. Accomplishments 01 Rapid detection and classification of foodborne pathogens using the BARDOT system. ARS researchers at Wyndmoor, Pennsylvania developed a novel biosensor-based method utilizing the BARDOT (Bacterial Rapid Detection using Optical Scattering Technology) system for high- throughput detection of Campylobacter in food. A comprehensive laser- scattering image library has been constructed by automatically scanning over 7000 bacterial colonies of different strains of C. jejuni, C. coli, Salmonella, E. coli O157:H7, and L. monocytogenes. Based on the image library, BARDOT was able to classify Campylobacter colonies and differentiate them from Salmonella, E. coli O157:H7, and L. monocytogenes. The application of BARDOT to ground chicken samples has been significantly improved by means of passive filtration to overcome the interferences from the food matrix. The rapid, high-throughput, automated, and nondestructive screening of Campylobacter colonies from food using BARDOT system has resulted in a publication and also assisted in the technology transfer from Purdue Research Foundation to Hettich (a German incubator/lab automation company).
Impacts
(N/A)
Publications
- He, Y., Reed, S.A., Bhunia, A.K., Gehring, A.G., Nguyen, L.T., Irwin, P.L. 2015. Rapid identification and classification of Campylobacter spp. using laser optical scattering technology. Food Microbiology. 47:28-35.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a na�ve library of biorecognition elements for bacterial typing-An alternative to �molecular typing.� Approach (from AD-416): This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/ or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations. Progress was made on all four objectives of this project (falls under National Program 108) by ARS researchers at Wyndmoor, PA with the goal to develop accurate methods for the detection and identification (ID) of pathogenic, foodborne bacteria. Successful detection/ID typically necessitates concentration via physical means (e.g., filtration) or increasing the number of factors (or biomarkers) through non-deleterious cell growth or genetic material amplification (i.e., PCR). For Objectives 1A and 1B, ground poultry has proven to be a much more challenging matrix than ground beef. Substantial amounts of solid material remain in suspension after filtration, and ground poultry solids were determined to be highly inhibitory to PCR detection. Treatment of the filtered ground poultry meat with detergents and enzymes was successful in dissolving much of the solid, and recovery of low levels (2 CFU/g) of E. coli, Salmonella and Listeria have been achieved. However, consistent results over multiple days/samples have been elusive. For Objective 2, the retention of Yersinia enterocolitica (YE) (and Yersinia pestis; YP) expression of virulence plasmid associated gene product, required for Congo Red binding, was demonstrated using a developed screening method. Also demonstrated was that low temp (28�C) culture growth of YE (&YP) was considerably more conducive to retention of virulence plasmid over higher temp (~37�C) conditions. For Objectives 3 & 4, we have initialized research using next generation sequencing (NGS) for genotyping and characterization of Campylobacter isolates from retail beef and chicken. A total of 10 Campylobacter strains including 7 C. jejuni and 3 C. coli were isolated from 17 independent meat packages. Genomes of all the isolates were subjected to NGS of barcoded libraries using the Ion Torrent platform. Comprehensive genomic sequence data of these isolates are being analyzed. Results will enhance our knowledge of two major species of Campylobacter as well as the genetic diversity of Campylobacter, especially with respect to pathogenesis, drug resistance, food environment, and transmission. For Objective 4, loop-mediated isothermal amplification (LAMP) is a relatively new method for detection of specific bacterial DNA targets. In contrast to PCR, LAMP uses a single, relatively low (60�C) temp, which simplifies construction of instruments promoting portability. In-house collaboration has yielded new LAMP assays for L. monocytogenes and Salmonella enteritidis that have been tested for specificity and sensitivity. The assays are specific for the target organism and provide detection limits of ~100 CFU per reaction. In addition for Objective 4, three protocols were developed to significantly shorten and improve the process of selecting DNA aptamers to bacterial targets. The first protocol employs real-time PCR with SYBR green detection for amplification of aptamer libraries. By monitoring amplification in real time, the optimum level of amplification can be selected in each step, avoiding poor yield or production of side-products resulting from under- or over-amplification. The second protocol provides separation and elution of purified single-stranded aptamer directly from the PCR amplification mixture. This eliminates the two purification steps normally used after PCR and after single-strand production, significantly shortening the process and reducing losses. The third protocol provides a rapid binding assay using 96 well filter plates. Careful choice of fluorescent label and wavelengths allows quantitation of low-level binding and determination of binding constants in minutes, without interference from bacteria or filter membrane. A dozen targets can be screened at once in less than 30 min. This approach can also be used for highly-parallel selection of aptamers against many target bacteria. Accomplishments 01 Rapid detection and classification of Campylobacter jejuni and coli using BARDOT. Through collaboration with scientists at Purdue University (West Lafayette, Indiana), ARS researchers at Wyndmoor, Pennsylvania have developed a laser-based scattering method called BARDOT (Bacterial Rapid Detection using Optical scattering Technology) for high-throughput screening of Campylobacter bacteria in food. Upon the completion of a Campylobacter laser scattering image database, BARDOT was able to automatically scan and classify a large number of Campylobacter jejuni and Campylobacter coli colonies with over 90% accuracy. Application of BARDOT in real food samples has been improved by using passive filtration to overcome the interferences from food matrix. Food regulators and producers may apply this technique for the identification of harmful pathogens. 02 Development of novel typing arrays for bacterial pathogens and toxins. Identification of bacteria requires methods that employ unique and selective agents (e.g., antisera) for classifying bacteria into distinct and recognizable groups or types (e.g., toxin type). Typing of pathogens is critical for preventing the spread of foodborne outbreaks. Rapid typing methods allow for timely epidemiological investigations that trace contagion back to its source. ARS researchers at Wyndmoor, Pennsylvania have developed two novel, high- throughput (handles large numbers of sample at once) antibody microarray techniques: 1) arrayed capture antibodies and a universal bacterial label with traditional fluorescence scanning and 2) a colorimetric sandwich ELISA (enzyme-linked immunosorbent assay) comprised of array-printed capture antibodies, sample target (e.g., Shiga toxin) �sandwiched� between the capture and enzyme-associated reporter antibodies, and very inexpensive office flatbed page scanning. The two rapid, ~2 hr, antibody-based microarray typing methods have been developed and applied to the simultaneous serotyping of various Shiga toxin-producing E. coli (STEC) and toxin typing of STECs induced to generate and release toxin during growth in the presence of low levels of a stress-inducing antibiotic. The former system has already found application for evaluation of typing antibodies produced by two companies and one university. The latter technique has been applied to evaluation of monoclonal antibodies produced by ARS researchers at Albany, California and may find wide use in field applications in large part due to its simplicity, high-throughput format, and low cost.
Impacts
(N/A)
Publications
- Bhaduri, S. 2014. Yersinia enterocolitica. Encyclopedia of Food Microbiology. In: Batt, C.A., Tortorello, M.L. (Eds.), Elsevier Ltd, Academic Press. Vol 1. pp. 838-848.
- He, Y., Xie, Y., Reed, S.A. 2013. Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates. In: Walker, John M. Methods in Molecular Biology. New York, NY:Humana Press. p. 103-111.
- Bhaduri, S., Sheen, S., Sommers, C.H. 2014. Radiation resistance and loss of crystal violet binding activity in Y. enterolitica suspended in raw ground pork exposed to gamma radiation and modified atmosphere. Journal of Food Science. 79:911-916.
- Gehring, A.G., He, X., Fratamico, P.M., Lee, J., Bagi, L.K., Brewster, J.D. , Paoli, G., He, Y., Xie, Y., Skinner, C.B., Barnett, C., Harris, D. 2014. A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins. Toxins. DOI: 10.3390/toxins6061855.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a na�ve library of biorecognition elements for bacterial typing-An alternative to �molecular typing.� Approach (from AD-416): This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/ or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations. Progress was made on all four objectives and their subobjectives, all of which fall under National Program 108, Component I, [Microbial] Pathogens, Toxins and [non-biological-based] Chemical Contaminants: subdivided in Pre-harvest and Post-harvest. Progress on this project focuses on Problem Statement 1.C, Technologies for the Detection and Characterization of Microbial Contaminants. Substantial progress was made on every subobjective outlined in this project plan. The goal of this project is to develop accurate methods for the detection and identification (ID) of pathogenic, foodborne bacteria. Successful detection/ID typically necessitates concentration via physical means (e.g. , filtration) or increasing the number of factors (or biomarkers) through non-deleterious cell growth or genetic material amplification (i.e., PCR). For Objective 1A, we have developed a fast method for separating and concentrating bacteria from foods using leukocyte reduction filters (commonly used in hospitals for blood component separation). For Objective 1B, we have optimized the extraction of unique DNA factors using in-house developed reagents. For Objective 2A, a simple, economical, and highly reliable test using select organic dyes was developed to promote the rapid detection and isolation of Yersinia spp. For Objective 3A, an antibody-based microarray platform was developed for the typing of �Big Six� Shigatoxin-producing E. coli. For Objective 3C, a database of scan patterns generated by the BARDOT (bacteria rapid detection using optical scattering technology) system was created for the rapid ID of Campylobacter spp., however, sporadic results for some Campylobacter spp. will improve with a newer version of BARDOT (recently acquired). Also, in collaboration with FDA, a multiplex qPCR method was developed for the detection of E. coli O157:H7, and Listeria monocytogenes in soft cheeses. When growing bacteria in a lab, it is impossible to use a rapid method (e.g., qPCR) to calculate the initial number of bacteria unless a Most Probable Number (MPN) technique is also used. qPCR-MPN was employed with Campylobacter spp., but we found that ca. � of the results were problematic due to the non-random distribution of the bacteria in fat-laden chicken wash samples. For Objective 4A, in- silico studies were conducted to assess the feasibility of the proposed Restriction Fragment Sequence Polymorphism (RFSP) method for typing pathogens. Several hundred commercially available restriction enzymes were assessed with ca. 100 sequences of organisms currently in the CDC�s PulseNet PFGE database. Discrimination between bacteria was poorer using RFSP than by PFGE. Therefore, further pursuit of the RFSP approach is not recommended. In-silico studies were also conducted on Double Restriction Fragment Length Polymorphism methods. Preliminary results indicate that this is a promising approach and that a few combinations of restriction enzyme pairs could provide discriminatory power comparable to or better than PFGE. Accomplishments 01 Developed methods for separating and concentrating pathogenic bacteria from various food matrices. Foods and harmless bacteria in foods can interfere with accurate detection of harmful pathogens. ARS researchers at Wyndmoor, Pennsylvania have developed rapid filtration and centrifugation methods that not only separate, but to also concentrate target pathogens (Escherichia, Salmonella, Listeria, and Campylobacter spp.) from various food matrices including ground poultry and beef. Food regulators and producers will benefit immensely from applying these techniques since testing for harmful pathogens would be streamlined in terms of expense and time. 02 Ensuring the ability to detect harmful Yersina pestis (YP) in food. Accurate detection of virulent YP requires YP to have an intact virulence plasmid (small, circular strand of DNA). However, YP may lose this plasmid if it is mistreated during growth enrichment and/or isolation. ARS researchers at Wyndmoor, Pennsylvania have developed methods that were used to study the stability of the plasmid during growth of YP in raw ground meats (pork and beef). Producers and regulators may use this information to reduce the incidence of YP in foods. 03 Developed a typing assay for Shiga-toxin producing E. coli (STEC) using microarrays. Methods are necessary for the rapid identification of detection and identification (typing) of harmful bacteria in foods. Antibody and DNA typing (identification) microarray platforms have been developed on both glass and relatively inexpensive polystyrene plastic substrates. ARS researchers at Wyndmoor, Pennsylvania have produced systems that, using either antibodies or computer-designed nucleic acid probes/primers, interrogate large numbers of samples for E. coli O157:H7 and/or the �Big Six� non-O157 STECs. Total assay times were typically under 3 h with real-time (non-growth enriched samples) with relatively low detection limits. The published finding may arm bacterial typing labs and regulatory testing agencies with additional means for ensuring biosafety as well as biosecurity of foods. 04 Developed an identification method for foodborne pathogens using BARDOT and RT-PCR. There is a need to replace current methods for the identification of harmful bacteria in foods since such testing often takes long days to weeks. Faster identification reduces has benefits ranging from shorter food product holding times to accelerated epidemiological investigations. ARS researchers at Wyndmoor, Pennsylvania have combined BARDOT (bacteria rapid detection using optical scattering technology; technology developed by our collaborators in the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana) with multiplex RT-PCR (real time PCR) for the prescreening and identification of identification of pathogens (Campylobacter jejuni and C. coli) in foods. Food regulators and producers may apply this technique for the identification of harmful pathogens.
Impacts
(N/A)
Publications
- Gehring, A.G., Barnett, C., Chu, T., Debroy, C., D'Souza, D., Eaker, S., Fratamico, P.M., Gillespie, B., Hedge, N., Jones, K., Lin, J., Oliver, S., Paoli, G., Perera, A., Uknalis, J. 2013. A high-throughput antibody-based microarray typing platform. Sensors. DOI: 10.3390/s130505737.
- Brewster, J.D., Paoli, G. 2013. DNA extraction protocol for rapid PCR detection of pathogenic bacteria. Analytical Biochemistry. 442(1):107-109
- Hegde, N.V., Praul, C., Gehring, A.G., Fratamico, P.M., Debroy, C. 2013. Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray. Journal of Microbiological Methods. 93(3):273-276.
- Paul, M., Van Hekken, D.L., Brewster, J.D. 2013. Detection and quantitation of Escherichia coli O157:H7 in raw milk by direct qPCR. International Dairy Journal. 32:53-60. Available:
- Gehring, A.G., Boyd, G., Brewster, J.D., Irwin, P.L., Thayer, D.W., Van Houten, L.J. 2012. Comparison of antibodies raised against heat-and gamma radiation-killed bacteria. Journal of Microbial and Biochemical Technology. DOI 10.4172/1948-5948.S2-004.
- Bhaduri, S., Phillips, J.G. 2013. Growth of a plasmid-bearing (pYV) Yersinia pestis KIM5 in retail raw ground pork. Foodborne Pathogens and Disease. 10:467-471.
- Suo, B., He, Y., Irwin, P.L., Gehring, A.G. 2013. Optimization and application of a custom microarray for the detection and genotyping of E. coli O157:H7 in fresh meat samples. Journal of Food Analytical Methods. 6:1477-1484.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a na�ve library of biorecognition elements for bacterial typing-An alternative to �molecular typing.� Approach (from AD-416): This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations. The goal of this project is to develop accurate methods for the detection and identification (ID) of pathogenic (harmful), foodborne bacteria. Prerequisite is the need to maintain the very factors/biomarkers/determinants that uniquely distinguish bacterial species/strains. Typically, successful detection/ID necessitates concentration via physical means (e.g., filtration) or increasing the number of factors through cell growth or a genetic material amplification process (PCR; polymerase chain reaction). Also, accurate detection/ID requires that any growth, isolation (e.g., from food samples), or manipulation does not alter the unique factors. Researchers at ARS in Wyndmoor have developed a fast method for separating and concentrating bacteria from foods using leukocyte removal filters (commonly used in hospitals for blood component separation). We have optimized the extraction of unique DNA factors using in-house developed reagents. A simple, economical, and highly reliable test using select organic dyes was developed to promote the rapid detection and isolation of the pathogenic bacteria, Yersinia species (spp.). In collaboration with scientists from Purdue University, a database of scan patterns generated by the BARDOT (bacteria rapid detection using optical scattering technology) system was created for the rapid ID of the pathogenic bacteria, Campylobacter spp., however, sporadic results for some Campylobacter spp. will improve with a newer version of BARDOT. In collaboration with FDA, a multiplex qPCR (quantitative PCR) method was developed for the detection of E. coli O157:H7, and Listeria monocytogenes in soft cheeses. With culture enrichment, quantitation of bacteria with a rapid method (e.g., qPCR) is impossible unless a technique known as MPN (most probable number�a quantitative technique based upon dilution to the point that no bacterial cells exist and therefore some diluted samples exhibit no growth� back calculation is performed to determine initial sample cell concentration) is concomitantly used. qPCR-MPN was employed with Campylobacter spp., but we found that approx. � of the results were problematic due to the non- random distribution of the bacteria in fat-laden chicken wash samples. In-silico studies were conducted to assess the feasibility of the proposed Restriction Fragment Sequence Polymorphism (RFSP) method for typing foodborne pathogens. Several hundred commercially available restriction enzymes (biomolecules that can cleave DNA, or RNA, at specific sequence locations) were assessed with approx. 100 sequences of organisms currently in the CDC�s PulseNet PFGE (pulsed-field gel electrophoresis) database. Discrimination between bacteria was poorer using RFSP than by PFGE. Therefore, further pursuit of the RFSP approach is not recommended. In-silico studies were also conducted on Double Restriction Fragment Length Polymorphism methods. Preliminary results indicate that this is a promising approach and that a few combinations of restriction enzyme pairs could provide discriminatory power comparable to or better than PFGE. Accomplishments 01 Development of improved foodborne bacteria detection methods via investigation of environmental barriers to bacterial growth and survival The toxicity of Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis bacteria depends on the presence of a small, circular piece of DNA inside the cell which is referred to as a virulence plasmid (pYV). The pYV is unstable in all three bacteria and if it lost during growth or processing, the bacteria may not be properly detected or identified. In order to better understand pYV stability, ARS researcher at Wyndmoor, Pennsylvania developed a procedure to monitor its presence Yersinia bacteria. The procedure exploits the low calcium response (Lcr and Congo red (CR) binding by the bacteria. The Lcr-CR positive bacteri (pYV-bearing strains) were used to study the growth of and to monitor th pYV stability of virulent Y. pestis and Y. pseudotuberculosis in raw ground beef. The published finding will arm regulatory testing agencies with additional means for ensuring biosafety as well as biosecurity of foods. 02 Identification of pathogenic Staphylococcus aureus strain by classification of toxin genes. Food producers and regulatory agencies ar in constant need of improved means for bacterial classification so that identification of harmful, bacterial contaminants in foods may be dealt with in a timely manner. It is of critical importance that such investigative methods be very accurate for tracing back the source for bacteria isolated from human samples (i.e., clinical isolates). In rece decades, such methods have compared genetic patterns of isolates against known bacteria. ARS researchers at Wyndmoor, Pennsylvania in conjunctio with collaborators at Shanghai Jiao Tong University (China), have compar three genetic classification or typing schemes for their ability to distinguish between pathogenic strains of S. aureus. The gene typing schemes were PFGE (pulsed-field gel electrophoresis), MLST (multilocus sequence typing), and a custom-developed method termed �Toxin Gene Typin Method.� In the analysis of over 100 S. aureus clinical isolates, three (of 18) known toxin genes were found to be most common and the Toxin Gen Typing Method was noted to give the best results. This study was published in a relatively high impact journal and the results can be use for clinical as well as food borne outbreak-related investigations.
Impacts
(N/A)
Publications
- Bhaduri, S. 2012. Modification of an acetone-sodium dodecyl sulfate disruption method for cellular protein extraction from neuropathogenic Clostridium botulinum. Foodborne Pathogens and Disease. 9:172-174.
- Bhaduri, S., Smith, J. 2011. Virulence plasmid (pYV)-associated expression of phenotypic virulent determinants in pathogenic Yersinia species: a convenient method for monitoring the presence of pYV under culture conditions and its application for isolation/detection of Yersinia pestis in food. Journal of Pathogens. DOI: 10.4061/2011/727313.
- Bhaduri, S., Phillips, J.G. 2010. GROWTH MODEL OF A PLASMID-BEARING VIRULENT STRAIN OF YERSINIA PSEUDOTUBERCULOSIS IN RAW GROUND BEEF. Zoonoses and Public Health. DOI: 10.1111/j.1863-2378.2009.01271.
- Xie, Y., He, Y., Gehring, A.G., Hu, Y., Tu, S., Shi, X. 2011. Genotypes and enterotoxin gene profiles of Staphylococcus aureus clinical isolates from China. PLoS One. DOI: 10.1371/journal.pone.0028276.
- Xie, Y., Xu, S., Hu, Y., Chen, W., He, Y., Shi, X. 2012. Rapid identification and classification of Staphylococcus aureus by attenuated total reflectance fourier transform infrared spectroscopy. Journal of Food Safety. 32(2):176-183.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a na�ve library of biorecognition elements for bacterial typing-An alternative to �molecular typing.� Approach (from AD-416) This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations. This project was going through the OSQR review process during FY 2011. Progress addressed by SYs working on this project are documented in the reports for 1935-42000-058-00D or 1935-42000-062-00D.
Impacts
(N/A)
Publications
- Bhaduri, S., Chaney, K.J., Smith, J.L. 2011. A procedure for monitoring the presence of the virulence plasmid (pYV) in Yersinia pestis under culture conditions. Foodborne Pathogens and Disease. 8:459-463.
- Bhaduri, S. 2011. Effect of salt and acidic pH on the stability of virulence plasmid (pYV) in Yersinia enterocolitica and expression of virulence-associated characteristics. Food Microbiology. 28:171-173.
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