1a. 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 naïve library of biorecognition elements for bacterial typing-An alternative to “molecular typing.”
1b. 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.
3. Progress Report:
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. ¼ 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.
1. 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 researchers at Wyndmoor, Pennsylvania developed a procedure to monitor its presence in Yersinia bacteria. 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. The published finding will arm regulatory testing agencies with additional means for ensuring biosafety as well as biosecurity of foods.
2. Identification of pathogenic Staphylococcus aureus strain by classification of toxin genes. Food producers and regulatory agencies are 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 recent decades, such methods have compared genetic patterns of isolates against known bacteria. ARS researchers at Wyndmoor, Pennsylvania in conjunction with collaborators at Shanghai Jiao Tong University (China), have compared 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 Typing 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 Gene Typing Method was noted to give the best results. This study was published in a relatively high impact journal and the results can be used for clinical as well as food borne outbreak-related investigations.
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.