Advances and Updates on Pathogen Test Methodology

Transcription

1 Advances and Updates on Pathogen Test Methodology Alex Brandt, Ph.D. Chief Science Officer Food Safety Net Services (FSNS) September 5, 2018 Pathogen Control and Regulatory Compliance in Beef Processing Conference

2 DISCLAIMERS Any technologies that are mentioned in this presentation are mentioned only to provide examples of methodological changes that are occurring in the testing industry and any mention of a technology does not constitute a promotion of that technology on the part of FSNS or any other party. This is a beef safety conference, but many methods that are currently being used for other food matrices may have crossover with beef safety; thus they are discussed within this presentation.

3 Overview Beef Trim Sampling Procedures Limits Testing for Salmonella The Race to Zero for Listeria Isolate-Free Molecular Subtyping MALDI-TOF Based Identification DNA Sequencing Updates Detection Using DNA Sequencing

4 Beef Trim Sampling Procedures Biggest changes going on in beef trim testing right now deal with using the USDA-ARS manual sampling device (MSD) and continuous sampling device (CSD) to replace N60 excision sampling and the IEH N60 Plus Sampler. N60 sampling process entails excising 60 1 x 3 samples of meat from the surface of beef trim pieces and are tested as 325 g or 375 g samples. IEH N60 Plus Sampler is a stainless steel sampling device on a drill that collects surface tissue (where contamination is located) and uses a smaller sample size than 375 g. Theoretically increases sensitivity since E. coli O157:H7 is only located on the surface of the meat, and the more surface tissue and less internal tissue you obtain the better. Credit: USDA FSIS Credit: IEH, Inc.

5 MSD and CSD Methods from ARS Rather than actually taking portions of meat as the sample to be tested, a piece of absorbent material made from cellulose can be used to sample the surface of the meat. Can be used in two different ways: 1. Continuous Sampling Device (CSD) - Placed on the end of a trim belt right before the pieces dump into a combo bin so that all pieces are sampled. 2. Manual Sampling Device (MSD) - Used to rub the top layers of trim in a combo bin to collect a sample from the same layers that an N60 sample would be collected from. Credit: Wheeler, et al.

6 Continuous Sampling Device (CSD) New technology no objection letter from FSIS. Three pieces: stainless steel base, two piece plastic cassette, sampling cloth. ARS has patent pending and has licensed to FREMONTA to produce the product called MicroTally Pros: In theory most every piece of trim is sampled, less labor intensive, less meat lost to sampling Cons: May fall off into combo bin creating foreign material issue, limits of absorbency, antimicrobial buildup and viability Credit: FSIS Credit: Wheeler, et al.

7 Manual Sampling Device (MSD) New technology no objection letter from FSIS. Just use the sampling cloth portion and rub it over the top of the meat in a combo bin using sterile gloves. ARS also has patent pending and also has licensed to FREMONTA to produce the product called MicroTally Pros: Still less labor intensive than excision sampling and less meat is used up for testing. No concerns about losing the cloth and creating foreign material issues Cons: Still only get the top few layers as opposed to what the CSD gets, limits of absorbency Prevalence of Natural E. coli O157:H7 by Sampling Method and Lean Type Credit: FSIS Lean Type N N60 Plus MSD 50% % 7.4% 80% % 2.3% Prevalence of Natural E. coli O157:H7 and Salmonella by Sampling Method (90% Lean) Credit: Wheeler, et al. Organism N N60 Plus MSD E. coli O157:H % 0% Salmonella % 4.9%

8 Future of MSD and CSD Many establishments have already generated internal data to validate the equivalency of these sampling methods with their historic N60 excision sampling and IEH N60 Plus Sampler methods. What is lagging is the full validation of rapid testing methods (and MLG procedures) to accommodate testing the CSD and MSD. Changes in enrichment media ratios, incubation times, etc. One test kit manufacturer has generated and made some data available. Others are still working on the validations. FSNS doing some internal validations. Future studies will look at validation of using a single cloth to sample multiple combos (meant to mimic a five combo lot of dry composited N60 samples today) and other compositing schemes. Predictions: Prevalence of E. coli O157:H7 may increase slightly among companies doing testing since the data have shown better recovery of the pathogen. Not sure how it will make a difference for Salmonella. Will certainly reduce labor and will reduce media usage at the lab level.

9 Limits Testing for Salmonella In the FSIS world, Salmonella is not an adulterant for raw meat and poultry. Still have to meet performance standards, but the finding of Salmonella in a raw product does not make the product outright adulterated. Prevalence rates can be fairly high, especially for products such as ground poultry, but does not mean that the concentrations in the products are necessarily high. Whether or not an individual develops salmonellosis is based on several factors such as the immune status of the individual, the amount of product consumed, and the concentration of Salmonella in the product. Concentration may be too low to cause disease. Some companies are taking the approach of trying to determine which of their product lots have concentrations of Salmonella that will pose a public health risk. Credit: OnHealth Credit: Food Safety News

10 Old Methods of Salmonella Quantitation MPN (Most Probable Number) testing, but that is very time consuming and costly. - Generally run a 3 X 3 or 3 X 5 setup with three to five large portions enriched and tested, three to five medium portions enriched and tested, and three to five small portions enriched and tested. - Thus have to run 9-15 tests on all of these tubes. - Take the number of tubes positive at each level and score this to come up with the concentration of Salmonella. Could do direct plating, but that may not pick up low concentrations, and there is a lot of background organism growth that will occur on the plates and cause inaccurate counts. - For instance using XLT4 and black colonies will allow things like Citrobacter and Proteus to be included in the count. Credit: Microbeonline.com Credit: Wikimedia

11 How Limits Testing Works Limits Testing bends the rules on using traditional assays that provide a binary outcome (i.e. positive/negative) by using them in a semi-quantitative manner. Most of those assays are just trying to determine if there is any Salmonella present (i.e. as low as 1 CFU/sample). Thus, they provide optimal enrichment incubation conditions and time to find any Salmonella. Not very useful if we expect a lot of it. But, if the incubation conditions are modified, such as by significantly shortening them, we may be able to only catch those samples that have high enough levels of Salmonella starting out (i.e. as high as 1 CFU/g) that will produce a positive result in a shorter and less than optimal enrichment incubation time. Credit: Combase.com

12 Limits Testing Concept Assay Detection Limit If I am looking for a starting concentration of 100 CFU/g in my product, I know I can pull it for testing at 2.5 hours and it should give me a positive, if I am consistent in my conditions every time.

13 Limits Testing Concept Assay Detection Limit Then, if I use an assay with a better detection limit (i.e. 3.5 log vs 4.0 log) I can get an answer even faster (i.e. 1.3 hours vs. 2.5 hours).

14 Limits Testing Publications IAFP 2015 poster investigated the use of the Roka Bioscience, Inc. Atlas Salmonella SEN Detection System, to find limits testing conditions that detect Salmonella concentrations of 0.5 CFU/g and 1 CFU/g in raw ground turkey. Phase I of the study evaluated how enrichment conditions could be optimized to consistently detect each of these levels of contamination. Phase II involved refining these enrichment conditions down to half hour intervals in order to tighten the range of the known starting concentration of Salmonella in the food product sample. Phase III of the study involved evaluation of ten samples for each set of conditions in triplicate studies to provide full validation of the different sets of conditions. Overall, the final outcome was that researchers were able to develop a limits testing based approach to accurately detect 1 CFU/g (their public health limit) of Salmonella in a 375 g ground turkey sample, in a short enough time that they could make a decision to divert product within the course of one shift.

15 Future of Limits Testing Concept of limits testing is likely to be used more in the future by the meat industry due to its ability to discern those lots of food products that are expected to have a negative public health impact. Will help those manufacturers justify the implementation of testing programs for those pathogens that are frequently present in products at concentrations that are low enough such that they are not likely to create a public health issue, but that can create a public health issue if their concentration spikes up in a product for some particular reason. Will also provide them with scientifically based justification not to have to treat testing programs for these pathogens as if they are zero tolerance based programs. Should likely help encourage manufacturers who currently avoid implementing testing programs for non zero tolerance pathogens to implement testing programs that will still allow them to catch problematic product lots, but also be able to release product lots that are not expected to be problematic. Operationally, this testing is also great because it is fast TAT to make a decision! Mostly used in poultry, but can this be used for certain beef products with traditionally high loads of non-adulterant pathogens like Salmonella?

16 The Race to Zero for Listeria Biggest change currently going on in Listeria testing is the race to zero enrichment time for testing on environmental sponge samples. Just take sponge/swab suspension liquid and test directly from the liquid. Traditional ELFA (which is nearly all testing we do in FSNS) and PCR assays take at least a day of incubation to have enough Listeria target present to have detection. Still in infantile stages, and needs lots of refinement, but there is a significant amount of movement in this area. Why zero enrichment time? If my amount of time to find the problem is short = my time to make a decision is short = my time to correct the problem is short = less product affected = less money lost. Can these methods be refined and used for other pathogens in beef processing carcass swabs for E. coli O157:H7, CSD and MSD samples for E. coli O157:H7?? Credit: 3M

17 Listeria Zero Enrichment Time One of the first assays on the scene that was zero enrichment time was the Sample6 Detect/L Assay. First seen around Now owned by IEH Laboratories and Consulting Group. Not 100% enrichment free, but is close. Add 6 ml of detection buffer to a sponge, squeeze by hand, and incubate at 30 C for 6 hours. After incubation, take 1 ml, centrifuge, take 300 µl, and add 300 µl of detection buffer. Then wait 1 min and put into the detector. Principle behind the assay: a cocktail of bacteriophages specific for Listeria are added to the sponge, attach to any Listeria cells there, inject DNA that codes for production of luciferase enzyme, which then builds up and can be detected when in contact with substrate. Credit: Sample6

18 Listeria Zero Enrichment Time Newest one available is Neogen ANSR Listeria Right Now (LRN). Claim to have a total of one hour of time to result. Detects very low numbers of Listeria spp., including L. monocytogenes, from environmental samples without enrichment. Employs an isothermal, amplified nucleic acid-based reaction to target rrna. Amplification occurs through a polymerization mechanism by a specific endonuclease. Detection occurs in real-time using a fluorescent molecular beacon. Ribosomal RNA is present in much greater numbers in Listeria cells than the traditional DNA target (~ ,000 copies per cell vs. 1 copy per cell for DNA). This can result in a 1,000 10,000 fold increase in target analyte concentration. The isothermal reaction within the instrument produces a constant cycle of molecular replication producing analyte copies much more quickly than traditional PCR reactions which run through a series of heating and cooling cycles. Credit: Neogen

19 Listeria Zero Enrichment Time Most recent movement has been to utilize the Roka Atlas Listeria Environmental Detection Assay as a zero enrichment time assay. Uses magnetic beads to capture Listeria specific rrna target material during extraction, performs transcription-mediated amplification to amplify over a billion fold, and performs probe detection with a chemiluminescent probe. Reads RLU and then calculates whether the sample is positive or negative. Because of the multitude of rrna copies in a single cell, one is able to detect very low numbers of cells on a sponge. Very similar concept to that of the Neogen LRN Assay. No specific data published to date, but this is being worked on. Credit: Roka Bioscience, Inc.

20 Listeria Zero Enrichment Time Future This is going to continue to gain traction as methods become more high throughput. Will help companies make decisions much faster about whether to clean, re-sample, and resume production or if further things like vector sampling need to be done. Will help make Listeria control much more real-time. However, lots of work to be done in this area yet as many of these assays are still very primitive and need a lot of refinement work to get them honed in correctly. Will require some re-thinking on the part of FSIS with regard to its Listeria guidance documents that give you a three day repeated testing scheme. May not need that anymore. Again, can this be used in other applications where we are doing surface sampling?

21 Traditional Molecular Subtyping Traditional methods of molecular subtyping have focused on determining relatedness of ISOLATES. Traditional methods include PFGE and Ribotyping. Both are like molecular barcodes for an ISOLATE to demonstrate relatedness. PFGE involves restriction enzyme digestion of total genomic DNA and running fragments on a gel. Ribotyping also uses restriction enzyme digestion of DNA, runs on membrane, and then tags 16S rrna gene DNA to make a pattern. Of course, genome sequencing is also always an option for this. More of this later in the presentation. Credit: Cornell University Credit: biomérieux

22 Isolate-Free Molecular Subtyping But what if you do not want to obtain an isolate, do not want to know the species via confirmation, and just want a molecular barcode (i.e. do not want to use WGS methods) for subtyping? Rheonix Listeria PatternAlert Assay enables users to detect molecular patterns in just six hours directly from a positive enriched sample, without the need for an isolate. Detects the presence or absence of independently occurring genetic targets that can sort Listeria into thousands of potential patterns. Credit: Rheonix

23 Isolate-Free Molecular Subtyping Each pattern generated by the assay encompasses a group of strains and may include multiple species of Listeria. The discriminatory power of the PatternAlert assay has been carefully calibrated to enable users to make informed decisions based on molecular patterns, without providing a strain level characterization equivalent to whole genome sequencing or pulsed field gel electrophoresis. With the Listeria PatternAlert assay, once a presumptive positive result has been obtained using an existing screening method, a sample from the initial enrichment can be processed directly on the Encompass Optimum workstation, without the need for an isolate. Rheonix Listeria Outputs

24 Isolate-Free Molecular Subtyping Future Already quickly gaining traction. Producers love the fact that they do not have to get an isolate from a traditional cultural confirmation process. Does not define the species for Listeria. Allows more regulatory flexibility but also allows you to source-track. This is quickly becoming competitive with the traditional methods for subtyping. None have allowed you to do any work from enrichments before. Will most certainly expand to other pathogens like Salmonella and Pathogenic E. coli.

25 Traditional Pathogen ID Methods Traditional pathogen identification methods are based on biochemical reactions whereby metabolic processes in an organism drive its identification procedures. Examples of traditional rapid biochemical profiling assays that are very widely used are API and VITEK.

26 Using MALDI-TOF for Pathogen ID Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI- TOF) Mass Spectrometry. Essentially a molecular scale Fast, easy and effective alternative for microbial ID. Has been validated against gold standard methods and is now FDA approved (2013) for clinical diagnostic applications for species level ID. Credit: Adapted from "What is Mass Spectrometry?" by Chace and Sparkman, ASMS, 2005

27 How Does Mass Spectrometry Work? Credit: Adapted from J. Prenni Identification and Characterization of Bacteria using Mass Spectrometry

28 How Does Mass Spectrometry Work? Credit: Adapted from J. Prenni Identification and Characterization of Bacteria using Mass Spectrometry and Bruker Daltonics

29 How Does Mass Spectrometry Work? Credit: Adapted from J. Prenni Identification and Characterization of Bacteria using Mass Spectrometry

30 Steps to Use MALDI-TOF for ID Step 1: Pick colonies from plates Step 2: Transfer colony to target and add matrix Step 3: Acquire spectral fingerprint. Can compare this spectral fingerprint to libraries of known cultures for ID.

31 Future for MALDI-TOF for ID Proven to be as accurate as gold standard methods Significant cost savings (time, reagent costs, technical expertise, etc.) FDA approved for clinical diagnostics (species level only) Commercially available as VITEK MS. Initial price of instrumentation is a deterrent, but could eventually replace traditional biochemical methods if it becomes cost effective and if good libraries are built. Could even possibly use for subtyping.

32 DNA Sequencing Updates Whole Genome Sequencing is obviously used heavily by the regulatory agencies (USDA and FDA) along with CDC to do source tracking of outbreaks and to replace other methods of subtyping such as PFGE and AMR testing. Metagenomics is being used heavily in research settings to track microbial population compositions in different sectors of production. Not much being done by meat industry with WGS (or any NGS technologies for that matter) due to uncertainty from FSIS about how the data will be handled. Last fall at WGS Public Meeting industry asked for safe harbors to allow them to generate and act upon their own data without regulatory intervention and database uploads to help alleviate this. Regardless, many advances in technology are currently occurring.

33 Central Dogma of Biology CENTRAL DOGMA OF BIOLOGY: DNA is transcribed to RNA. RNA is translated to Proteins. DNA is double-stranded and is comprised of four bases: A = Adenine T = Thymine C = Cytosine G = Guanine The sequence of the bases in DNA and RNA determines the amino acid sequence of the proteins that are used to build living organisms. DNA sequences can be used to determine relatedness of organisms. Sequence Similarity = Organism Relatedness

34 First Generation Sequencing The first generation of DNA sequencing technologies consisted of the Sanger chain termination method. Very labor intensive as it required manually reading gels. Eventually was refined for automated reads, but still not very efficient in generating large amounts of data. Was used for the Human Genome Project that began in Took 13 years to sequence the entire 3.2 Gb human genome.

35 Second Generation Sequencing Next generation of DNA sequencing came about with the invention of pyrosequencing (Roche 454). Allowed for mass parallelization of DNA sequencing reactions and bp reads. Further improvements came with the invention of Solexa/Illumina sequencing, which replaced beadbased emulsion PCR with DNA bound in a solid state to flowcells. Heavy investments from government agencies. These technologies are still sequence-by-synthesis technologies. This is where we have been for several years now and where we are at currently.

36 Third Generation Sequencing Mostly defined by the capability of single molecule sequencing (SMS), negating the requirement for DNA amplification like previous methods. Oxford Nanopore Technologies GridION and MinION are the most promising of new technologies. Are based on passage of the DNA molecule through a channel that blocks ion flow, thereby decreasing the current for a length of time proportional to nucleic acid length. This is where fastest development is currently taking place.

37 DNA Sequencing Updates Right now, FSNS sees DNA sequencing used most in event day tracking when a plant experiences a multitude of E. coli O157:H7 positives. Used as part of the investigation to link the strains that are isolated from over the course of the production day. People are still requesting PFGE a lot due to the very limited amount of data that comes from that process; essentially just a molecular barcode. More traction being gained for using metagenomics to assess spoilage organism populations over time. Will help predict shelf life better and will help determine which organisms are the ones contributing to shelf life issues. Lots of issues with blown packs, etc. can be resolved by doing metagenomics on the sample, finding the causative organism, and then tracking down the organism. Credit: Clear Labs, Inc.

38 DNA Sequencing Future Really depends on how the regulators give safe harbors to industry to use the technology without having every sequence taken and put into GenomeTrakr. Need to allow the industry to utilize it to do source tracking in plants to determine sources of persistence or sources of constant introduction of pathogens without all of the sequences being uploaded. Need to make sure there is good epidemiological metadata associated with sequences placed into GenomeTrakr. Should not make connections just based on sequence information. Need to have good metadata tying cases together or tying a food to a case. Industry really should push to get some traction on this. Why would we not use a method to track pathogens in our processes that the regulators are using? Why would we not want to be at the same level?

39 Using DNA Sequencing for Detection Traditionally we have used ELISA, PCR, and isothermal amplification to detect foodborne pathogens in enrichments. Clear Labs Clear Safety is the first automated NGS platform that s purpose-built for food safety testing. Uses the Oxford Nanopore Sequencer as the core detection device. Generates millions of data points per analysis that can be used to learn more than what can be obtained from regular screening methods. Provides data without the liability of whole genome sequencing.

40 Clear Safety Advantages Claims to provide molecular characterization of pathogens, but only provides the information needed for a particular program without liability of whole genome sequencing. For example, can choose to drill down to serotypes and strains based on the application needed. Can do serotype within 24 hours. Can efficiently screen an array of pathogens, increasing throughput and reducing costs. Enables one to run pathogen samples in parallel. Automated workflow that includes lysis, DNA extraction, library preparation, automated workflow for loading library onto the sequencer, and DNA sequencing Claim to provide >99.9% accuracy due to the fact that the specific sequence data for an entire genetic target or set of genetic targets is determined not like PCR or isothermal amplification, which only has primer and probe sequences in a genetic target.

41 Clear Safety Disadvantages Sample enrichment and extra processing time on the sequencer put it at a major disadvantage to PCR, which uses short enrichments and fast detection. Can get very costly to run unless you fully load the flow cells in the Oxford Nanopore Sequencer. Thus, you have to wait and batch to get optimal use of the instrument. PCR is much more inexpensive to generate a positive or negative result. Clear Labs has said that they will reimburse the costs of the flow cells to the end user if not fully optimized for maximum amount of samples. Only charge a per sample price to end user. Not sure how long that will last. Still, what happens with all of the data that is generated but not analyzed for the assay?

42 Future of DNA Sequencing for Detection Very novel concept. No one else is doing this currently. Eventually this could become very competitive with PCR and isothermal amplification methods if the reagents can become cheaper and the TAT can be made faster. Biggest holdups right now are cost and TAT. Can generate a lot of data about a pathogen in a sample enrichment without a lot of extra work. But also need to figure out how to discard data not wanted/needed. Could be the new detection method basis of the future, but definitely needs some refinements before then.

43 THANK YOU! QUESTIONS?