Summary
New microbial detection approaches utilizing whole genome sequencing are being increasingly applied for tracing microbial contaminants entering the food chain. The produce industry can directly benefit from powerful new methods such as shotgun metagenomics, which allows for the rapid identification of all the bacterial, viral, fungal, and protozoan pathogens in irrigation water, soil, or food samples in a single test. Furthermore, whole genome sequencing technologies are quickly becoming less expensive, and compact sequencing technologies like the Oxford Nanopore MinION device could potentially allow testing directly on-site in produce fields or other processing facilities for food safety surveillance programs. However, the application of these new whole genome sequencing technologies and approaches need to be verified and validated for use by the produce industry. The goal of this project is to investigate two technologies that offer slightly different approaches for pathogen detection, to identify the benefits and limitations of each, verify the results, and validate their applications by the produce industry for use in rapid pathogen detection in agricultural waters. The results of this study will provide recommendations, protocols and guidelines to the produce industry regarding the proper implementation of these technologies for pathogen surveillance.
Technical Abstract
New microbial detection approaches such as next generation sequencing (NGS) are being increasingly applied for tracing microbial contaminants entering the food chain. The produce industry can directly benefit from these powerful new approaches, such as shotgun metagenomics, which allow for the rapid identification of all the bacterial, viral, fungal, and protozoan pathogens in water, soil, or product samples in a single assay, thereby eliminating the need for many different detection assays. Ultimately, these novel approaches will be able to reduce the time and cost of not just food safety surveillance but also plant pathogen surveillance programs by combining everything into a single rapid assay. However, the application of these new NGS approaches need to be verified and validated for use by the produce industry. The goal of this project is to investigate two NGS technologies (Illumina iSeq100 and Oxford Nanopore MinION) that offer slightly different approaches for pathogen detection, and identify the benefits and limitations of each, verify the results, and validate the technologies for use by the produce industry. To accomplish this project, we have the following objectives: (1) Evaluate the detection limits of the iSeq100 and MinION sequencing technologies for three bacterial pathogens, two viral pathogens, and one protozoan pathogen in agricultural waters of varying quality. (2) Use shotgun metagenomics to characterize the microbial communities of agricultural waters from several Southwest regions using the “gold standard” of large amounts of Illumina sequencing and compare to the portable MinION technology. (3) Conduct whole genome sequencing, shotgun metagenomic, MinION and iSeq100 workshops/trainings for the produce industry. The results of this study will provide recommendations and guidelines to the produce industry regarding the proper implementation of iSeq100 and MinION technologies in food safety surveillance programs.
Research Objectives
We hypothesize that shotgun metagenomics without pathogen enrichment culture using either the Illumina iSeq100 or Oxford Nanopore MinION sequencing technologies can identify various pathogens alone or in combination in different qualities of agricultural water at the level of <104 cells/ml in a sample. The project has three objectives:
1. Evaluate the detection limits of the iSeq100 and MinION sequencing technologies for three bacterial pathogens, two viral pathogens, and one protozoan pathogen in agricultural waters of varying quality.
2. Use shotgun metagenomics to characterize the microbial communities of agricultural waters from several Southwest regions using the “gold standard” of large amounts of Illumina sequencing and compare to the portable MinION technology. 3. Conduct whole genome sequencing, shotgun metagenomic, MinION and iSeq100 workshops/trainings for the produce industry.
Findings & Recommendations
This study demonstrated the power of shotgun metagenomics to reduce sampling turnaround times by potentially eliminating the need for enrichments and other pathogen confirmation requirements. There are still significant hurdles that need to be overcome before it can be effectively applied to a company’s food safety program for routine surveillance/testing of agricultural water or other types of samples. Below are the critical findings and the needed steps to move the process closer to a usable technology/tool for industry:
Positive Results:
• A sequencing depth of 1,000 Mb/1.0 Gb or approximately 500,000 ONT MinION sequencing reads was effective at identifying all tested bacterial pathogens (Salmonella, L. monocytogenes, and E. coli O157:H7) in irrigation water samples at 102 CFU/ml or 100,000 CFU/L.
• Shotgun metagenomics using ONT MinION device was highly effective at identifying multiple pathogens in a single assay, meaning a single sample can be processed once for all bacterial foodborne pathogens reducing costs and time.
• ONT MinION device can effectively confirm the presence of a pathogen in a sample in less than 96 hours total without culture enrichment using shotgun metagenomics. Shotgun metagenomics using ONT MinION effectively detected the protozoan pathogen (Cryptosporidium parvum) in irrigation water samples at 102 oocysts/ml or 10,000 oocysts/L, but methods for DNA extraction were critical to effectively detect C. parvum oocysts in the samples particularly at lower concentrations.
• Location of the irrigation water sample did not impact the ability to detect bacterial or protozoan pathogens using shotgun metagenomics, therefore indicating that this technology/tool has the potential to work in other growing regions.
Negative Results:
• Shotgun metagenomics failed to detect the selected viral pathogens and will thus require additional methods in the sample processing to effectively confirm the presence of viral foodborne pathogens.
• Cryptosporidium requires specialized DNA extraction methods to effectively be detected using shotgun metagenomics, which limits the ability to rapidly detect this specific pathogen with other types of foodborne pathogens.
• Illumina iSeq100 technology could effectively detect pathogens using shotgun metagenomics similar to ONT MinION, but the amount of sequencing needed to detect at even 104 CFU/ml would be 5–10x higher than for ONT MinION.
• The current detection limit of 102 CFU/ml or 100,000 CFU/L for bacterial foodborne pathogens using shotgun metagenomics is 3 or 4 logs higher than is required to meet real world application demands by industry. Nevertheless, improved concentration methods could help to lower this detection limit.
• Higher turbidity levels caused issues with the ability to detect any foodborne pathogens, although at >200 NTUs, these turbidity levels were significantly higher than typically seen in agricultural water.
• Based on ATCC positive control experiments, there were differences in the two sequencing technologies’ ability to identify the abundance of various bacteria in irrigation water microbiome analysis samples, which raises concerns about this technology/tool, but does not appear to impact foodborne pathogen detection.
Next Step Recommendations:
• Develop methods that can reduce the detection limit to real world levels (<10 CFU/L) for bacterial foodborne and protozoan pathogens.
• Combine DNA extraction method/protocols for effectively extracting DNA from foodborne coccidia (e.g. Cryptosporidium, Cyclospora, Toxoplasma) and bacterial foodborne pathogens (e.g. Salmonella, Listeria, E. coli O157:H7, etc.) simultaneously to maximize the detection efficiency of these pathogens.
• Create a standardized method that will effectively identify any viral foodborne pathogens from agricultural water and can be integrated into a system with other types of foodborne pathogens to allow shotgun metagenomics to identify all types of foodborne pathogens from a single sample.
• Establish the exact number of sequencing reads/sequencing depth required to accurately identify any foodborne pathogen in an agricultural water sample at <10 CFU/L or lower every single time.
• Build a standardized computational pipeline that is simply to use to conduct the analysis needed to identify any foodborne pathogen from an agricultural water sample.
• Determine components of high-water turbidity that interfere with pathogen detection and generate standardized methods to overcome them. Explore the benefits of targeted sequencing technologies for deeper and better uniformity of coverage that can lead to greater analytical sensitivity, particularly for detection of foodborne viruses. The ONT MinION device is handheld and portable for on-site testing of agricultural water or other sample types for foodborne pathogens, however it will probably never be directly employed by growers or other industry representatives. It can still be easily utilized by small local testing laboratories or others that currently conduct frequent pathogen testing for industry, but all the above-mentioned next steps should be addressed before it is utilized for testing samples as part of a food safety program. However, industry representatives should still understand the capabilities and limitations to understand how to best apply this tool to their company’s food safety program when it does become feasible.