Project

Sequencing the supply chain for food safety and public health

Goal: Determine hazards that are transmitted via the food supply chain that begins at the farm and ends in consumption. The focus is to establish genomic techniques that enable culture independent methods via metaRNAseq to find pathogenic bacteria.

Methods: RNA Sequencing, Next Generation Sequencing, Big Data Analysis

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Bart C Weimer
added a research item
Background Antibiotic resistance genes (ARGs) are considered to be emerging environmental contaminants of concern potentially posing risks to human and animal health, and this research studied the prevalence of antimicrobial resistance in dairy manure. Methods This study is focused on investigating prevalence of ARGs in California dairy farm manure under current common different manure management. A total of 33 manure samples were collected from multiple manure treatment conditions: (1) flushed manure (FM), (2) fresh pile (FP), (3) compost pile (CP), (4) primary lagoon (PL), and (5) secondary lagoon (SL). After DNA extraction, all fecal samples were screened by PCR for the presence of eight ARGs: four sulfonamide ARGs ( sulI , sulII , sulIII , sulA ), two tetracycline ARGs ( tetW , tetO ), two macrolide-lincosamide-streptogramin B (MLS B ) ARGs ( ermB , ermF ). Samples were also screened for two mobile genetic elements (MGEs) ( intI1 , tnpA ), which are responsible for dissemination of ARGs. Quantitative PCR was then used to screen all samples for five ARGs ( sulII , tetW , ermF , tnpA and intI1 ). Results Prevalence of genes varied among sample types, but all genes were detectable in different manure types. Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW ( p = 0.01). Besides, network analysis indicated that sulII was associated with tnpA ( p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts ( p < 0.05). This research indicated current different manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs. Improvement of different manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.
Bart C Weimer
added a research item
In this work, we hypothesized that shifts in the food microbiome can be used as an indicator of unexpected contaminants or environmental changes. To test this hypothesis, we sequenced the total RNA of 31 high protein powder (HPP) samples of poultry meal pet food ingredients. We developed a microbiome analysis pipeline employing a key eukaryotic matrix filtering step that improved microbe detection specificity to >99.96% during in silico validation. The pipeline identified 119 microbial genera per HPP sample on average with 65 genera present in all samples. The most abundant of these were Bacteroides, Clostridium, Lactococcus, Aeromonas , and Citrobacter . We also observed shifts in the microbial community corresponding to ingredient composition differences. When comparing culture-based results for Salmonella with total RNA sequencing, we found that Salmonella growth did not correlate with multiple sequence analyses. We conclude that microbiome sequencing is useful to characterize complex food microbial communities, while additional work is required for predicting specific species’ viability from total RNA sequencing.
Bart C Weimer
added 2 research items
Poultry litter is used as soil amendment or organic fertilizer. While poultry litter is enriched with organic matter suitable for land, the presence of pathogens such as Salmonella in poultry litter is a concern. To investigate the effect of gaseous ozone on pathogen reductions in poultry litter, this study conducted a series of experiments that involved understanding of Salmonella Typhimurium and Escherichia coli O157:H7 inactivation at various doses of Ozone (O3) in wet and dry poultry litter conditions. Previously, ozone treatment has been shown to disinfect the surface of foods and plant materials including fruits, juices, and wastewater, however, additional research are needed to better understand the impacts of ozone on treatment of soil amendments. Sanitizing methods capable of eliminating pathogens of soil amendments are crucial to mitigate disease outbreaks related with litter/manure-based fertilizers. In this study, a bench scale continuous ozone treatment system was designed to produce O3 gas, with a range O3 concentrations (7.15–132.46 mg·L⁻¹), monitor ozone concentrations continuously, and control the ozone exposure time (15 to 90 mins) to understand the effectiveness of O3 in eliminating S. Typhimurium and E. coli O157:H7 in poultry litter. Results showed that 7.15 mg·L⁻¹ did not reduce the counts of S. Typhimurium until exposure to O3 for 90 min. The O3 concentrations of 43.26 ~ 132.46 mg·L⁻¹ exposure reduced the bacterial counts. Furthermore, the moisture content of poultry litter was found to be an influencing factor for pathogen reduction. The pathogen reduction rates were reduced when the moisture content was increased. At higher moisture content, high concentrations of O3 (132.46 mg·L⁻¹) were needed for pathogen reductions. The moisture content of 30% or lower was found to be more effective for controlling pathogen levels in poultry litter. Our study demonstrates that gaseous O3 treatment could be used as an additional decontamination technique to ensure the certain degree of microbiological safety of poultry litter based soil amendment.
Antibiotic resistance genes (ARGs) are emerging environmental contaminants of concern to both human and animal health. Dairy manure is considered reservoir of ARGs. This study is focused on investigating prevalence of ARGs in California dairy farm manure under current common manure management. A total of 33 manure samples were collected from multiple manure treatment conditions: 1) flushed manure (FM), 2) fresh pile (FP), 3) compost pile (CP), 4) primary lagoon (PL), and 5) secondary lagoon (SL). After DNA extraction, all fecal samples were screened by PCR for the presence of 8 ARGs: four sulfonamide ARGs ( sulI , sulII , sulIII , sulA ), two tetracycline ARGs ( tetW , tetO ), two macrolide-lincosamide-streptogramin B (MLS B ) ARGs ( ermB , ermF ). Samples were also screened for two mobile genetic elements (MGEs) ( intI1 , tnpA ), which are responsible for dissemination of ARGs. Quantitative PCR was then used to screen all samples for five ARGs ( sulII , tetW , ermF , tnpA and intI1 ). Prevalence of genes varied among sample types, but all genes were detectable in different manure types. Results showed that liquid-solid separation, piling, and lagoon conditions had limited effects on reducing ARGs and MGEs, and the effect was only found significant on tetW ( p = 0.01). Besides, network analysis indicated that sulII was associated with tnpA ( p < 0.05), and Psychrobacter and Pseudomonas as opportunistic human pathogens, were potential ARG/MGE hosts ( p < 0.05). This research indicated current manure management practices in California dairy farms has limited effects on reducing ARGs and MGEs. Improvement of manure management in dairy farms is thus important to mitigate dissemination of ARGs into the environment.
Bart C Weimer
added a research item
Animal proteins are essential elements of human and animal feed chain and improving the safety of human and animal feed requires understanding and controlling of the transmission of infectious agents in food chain. Many pathogenic infectious agents, such as prion protein is known to damage the central nervous system in the cattle. Bovine spongiform encephalopathy (BSE) results from infection agent (prion), and affects number of species such as cattle, human, and cats. In addition, Salmonella, pathogenic E. coli O157:H7, and Listeria monocytogenes were found in animal by-products used in the human and animal feed production. Increased interest in controlling microbial risks in human and animal feed is evidenced by a large number of publications, which highlights the need for examining the animal disposal method such as rendering process and provides a broader perspective of rendering process. While existing practices help greatly in controlling microbial contamination, this overview study showed that additional biosafety measures are necessary to ensure microbial safety in animal feed.
Bart C Weimer
added a research item
Here we propose that using shotgun sequencing to examine food leads to accurate authentication of ingredients and detection of contaminants. To demonstrate this, we developed a bioinformatic pipeline, FASER (Food Authentication from SEquencing Reads), designed to resolve the relative composition of mixtures of eukaryotic species using RNA or DNA sequencing. Our comprehensive database includes >6000 plants and animals that may be present in food. FASER accurately identified eukaryotic species with 0.4% median absolute difference between observed and expected proportions on sequence data from various sources including sausage meat, plants, and fish. FASER was applied to 31 high protein powder raw factory ingredient total RNA samples. The samples mostly contained the expected source ingredient, chicken, while three samples unexpectedly contained pork and beef. Our results demonstrate that DNA/RNA sequencing of food ingredients, combined with a robust analysis, can be used to find contaminants and authenticate food ingredients in a single assay.
James H Kaufman
added a research item
The rapid growth in biological sequence data is revolutionizing our understanding of genotypic diversity and challenging conventional approaches to informatics. With the increasing availability of genomic data, traditional bioinformatic tools require substantial computational time and the creation of ever-larger indices each time a researcher seeks to gain insight from the data. To address these challenges, we pre-computed important relationships between biological entities spanning the Central Dogma of Molecular Biology and captured this information in a relational database. The database can be queried across hundreds of millions of entities and returns results in a fraction of the time required by traditional methods. In this paper, we describe \textit{Functional Genomics Platform} (formerly known as OMXWare), a comprehensive database relating genotype to phenotype for bacterial life. Continually updated, the Functional Genomics Platform today contains data derived from 200,000 curated, self-consistently assembled genomes. The database stores functional data for over 68 million genes, 52 million proteins, and 239 million domains with associated biological activity annotations from Gene Ontology, KEGG, MetaCyc, and Reactome. The Functional Genomics Platform maps all of the many-to-many connections between each biological entity including the originating genome, gene, protein, and protein domain. Various microbial studies, from infectious disease to environmental health, can benefit from the rich data and connections. We describe the data selection, the pipeline to create and update the Functional Genomics Platform, and the developer tools (Python SDK and REST APIs) which allow researchers to efficiently study microbial life at scale.
James H Kaufman
added a research item
The diversity revealed by large scale genomics in microbiology is calling into question long held beliefs about genome stability, evolutionary rate, even the definition of a species. MacArthur and Wilson's theory of insular biogeography provides an explanation for the diversity of macroscopic animal and plant species as a consequence of the associated hierarchical web of species interdependence. We report a large scale study of microbial diversity that reveals that the cumulative number of genes discovered increases with the number of genomes studied as a simple power law. This result is demonstrated for three different genera comparing over 15,000 isolates. We show that this power law is formally related to the MacArthur-Wilson exponent, suggesting the emerging diversity of microbial genotypes arises because the scale independent behavior first reported by MacArthur and Wilson extends down to the scale of microbes and their genes. Assessing the depth of available whole genome sequences implies a dynamically changing core genome, suggesting that traditional taxonomic classifications should be replaced with a quasispecies model that captures the diversity and dynamic exchange of genes. We report Species population "clouds" in a defined microbiome, with scale invariance extending down to the level of single-nucleotide polymorphisms (SNPs).
James H Kaufman
added a research item
Foodborne disease outbreaks of recent years demonstrate that due to increasingly interconnected supply chains these type of crisis situations have the potential to affect thousands of people, leading to significant healthcare costs, loss of revenue for food companies, and-in the worst cases-death. When a disease outbreak is detected, identifying the contaminated food quickly is vital to minimize suffering and limit economic losses. Here we present a likelihood-based approach that has the potential to accelerate the time needed to identify possibly contaminated food products, which is based on exploitation of food products sales data and the distribution of foodborne illness case reports. Using a real world food sales data set and artificially generated outbreak scenarios, we show that this method performs very well for contamination scenarios originating from a single "guilty" food product. As it is neither always possible nor necessary to identify the single offending product, the method has been extended such that it can be used as a binary classifier. With this extension it is possible to generate a set of potentially "guilty" products that contains the real outbreak source with very high accuracy. Furthermore we explore the patterns of food distributions that lead to "hard-to-identify" foods, the possibility of identifying these food groups a priori, and the extent to which the likelihood-based method can be used to quantify uncertainty. We find that high spatial correlation of sales data between products may be a useful indicator for "hard-to-identify" products.
Bart C Weimer
added a research item
Importance: The development of antibiotic resistance threatens our modern medical achievements. The dissemination of antibiotic resistance can be largely attributed to the transfer of bacterial mobile antibiotic resistance genes (ARGs). Revealing the transfer network of these genes in bacteria and the forces driving the gene flow is of great importance for controlling and predicting the emergence of antibiotic resistance in the clinic. Here, by analyzing tens of thousands of bacterial genomes and millions of human and animal gut bacterial genes, we reveal that the transfer of mobile ARGs is mainly controlled by bacterial phylogeny, but under ecological constraints. We also found that dozens of ARGs are transferred between the human and animal gut and human pathogens. This work is the first demonstration of the whole profile of mobile ARGs and their transfer network in bacteria and provides further insight into the evolution and spread of antibiotic resistance in the nature.
Bart C Weimer
added 6 research items
The techniques of microbe community genome sequencing as applied to environmental samples - metagenomics - offer powerful insight into microbial community structure and ecology that can affect food safety decisions for public health security. In this paper, the design and characteristics of a new informatics service, the Metagenomics Computation and Analytics Workbench (MCAW), are presented and illustrated with reference to the analysis of metagenomics data. The service is designed to meet the requirements for analyzing metagenomic and metatranscriptomic sequence data to assess microbial hazards and food authentication in the supply chain. Moreover, MCAW provides for reliable storage and management of raw genomic sequences and analysis results, high-volume informatics processing, meticulous tracking of data provenance and processing steps, and function-rich visualization of results.
Bart C Weimer
added a project goal
Determine hazards that are transmitted via the food supply chain that begins at the farm and ends in consumption. The focus is to establish genomic techniques that enable culture independent methods via metaRNAseq to find pathogenic bacteria.