Category Archives: WGS

Research – Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States

Journal of Food Protection

This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

Research – New Challenges for Detection and Control of Foodborne Pathogens: From Tools to People


Contamination of foods by human pathogenic microorganisms is a major concern to both food safety and public health. The changes in consumers’ demand, the globalization of the food trade, and the progress on food production practices and processing technologies all pose new challenges for food industries and regulatory agencies to ensure the safety in food products.
With regard to microbiological safety, bacteria and viruses are the most common foodborne pathogens associated with both sporadic cases and outbreaks.
However, bacterial and viral microorganisms differ in terms of their behaviour in food matrices, their stability in food-related environments (e.g., food-contact surfaces, irrigating and processing waters), and their response to food processing technologies and controlling measures. Current methods do not meet all relevant criteria for effective monitoring plans, the main limitations being their sensitivity, the high workload and time requirement, and the inability to differentiate between viable and non-viable microorganisms. Thus, specific and sensitive methods need to be developed for their detection and quantification in com-plex matrices, such as food, for tracking their occurrence along the food chain to determine the sources of contamination, and for ultimately estimating the risk for consumers.
To fill these gaps, this Special Issue comprises four original research articles and are view paper focusing on the implementation of novel analytical techniques and approaches to foodborne pathogens along the food chain.
Zand and colleagues [1] reviewed the most recent advances of the application of flowcytometry (FCM) and fluorescence in situ hybridization (FISH) for the rapid detection and characterization of microbial contamination. FCM allows for a culture-independent quantification of microbial cells, also providing information on their physiological and structural characteristics which are relevant to assess their viability status. FISH is a nucleic acid-based method mainly applied in the medical and diagnostic fields. While FCM has been successfully used to detect and monitor microorganisms in water, state-of-the-art FCM and FISH protocols for food matrices still show significant limitations. The main pitfalls include complex sample preparation steps; the use of toxic substances; their limits of detection, especially for FISH assays; and the equipment price. Because of all these aspects, FCM and FISH have not yet gained considerable interest in food safety area for the detection of microbial pathogens. Future studies should focus on potential optimisation strategies for FCM and FISH protocols in food samples and their validation, as well as on the development of automated lab-on-chip solutions.
Moving to explore next-generation sequencing (NGS) applications in the produce industry, Truchado et al. [2] contributed to identify potential contamination niches of Listeria monocytogenes in a frozen vegetable processing plant. NGS is a sequencing technology that offers ultra-high throughput, a scalable and fast technique that allows the authors to characterize the isolates by a whole-genome sequencing (WGS) of 3multi locus sequence typing (MLST). The WGS analysis revealed the presence of four different sequence types (ST) contaminating 18% of the samples, including food contact surfaces (FCS), non-food contact surfaces (n-FCS), and final product. These ST were further classified into four different virulence types (VT) according to multi-virulence locus sequence typing (MVLST). Interestingly, an isolate detected in non-food-contact surfaces(n-FCS) also contaminated the final product, highlighting the relevant role of n-FCS as reservoir of L.monocytogenes that reached the final product.
Staphylococcus aureus is a foodborne pathogen considered to be one of the etiological agents of food-related disease outbreaks. Leng et al. [3] supported this Special Issue with a study on its control using the skin mucus extract of Channa argus as a source of antimicrobial compounds. Of interest, untargeted metabolomics were applied to decipherits antibacterial mechanism against S. aureus. Results indicated that the extract had great inhibitory action on its growth by inducing the tricarboxylic acid cycle and amino acid biosynthesis, which are the primary metabolic pathways that affect the normal physiological functions of biofilms.
The present collection includes a second contribution on the control of S. aureus authored by Kim and colleagues [4] who developed a real-time PCR method (qPCR) for the rapid detection and quantification of pathogenic Staphylococcus species.
Four specific molecular targets were identified based on pan-genome analysis, and results showed 100% specificity for 100 non-target reference strains with a detection limit as low as 102CFU/mL. Thus, the proposed method allows an accurate and rapid monitoring of Staphylococcus species and may help control staphylococcal contamination of food.
Moving to human viral pathogens, Macaluso et al. [5] reported the results of an investigation aimed to characterize the occurrence of human enteric viruses in shellfish, a food item with relevant risk for consumers. The study included data collected over two years on the prevalence of enteric virus contamination along the shellfish production and distribution chain in Sicily, Italy. The findings based on quantitative reverse transcription polymerase chain reactions (RT-qPCRs), as gold-standard molecular technique, showed that almost 6% of samples were contaminated with at least one enteric virus such as norovirus, hepatitis A virus, and/or hepatitis E virus. The origin of contaminated shellfish was traced back to Spain and several municipalities in Italy. Such contribution highlights the relevance
of routine monitoring programs to prevent foodborne transmission of enteric viruses and
preserve the health of consumers.
In summary, this Special Issue compile several contributions focused on novel technologies, approaches, and strategies demonstrated to be effective in controlling microbial contamination in food. All the articles provide valuable information to monitor and/or reduce contamination in food, food industry settings, and along the food chain. On a final note, the collection emphasizes the relevance of ensuring food safety and limiting the risk of microbiological contamination along the food chain to protect consumers.

Research – Integration of genomics in surveillance and risk assessment for outbreak investigation


Keeping food safe is a challenge that needs continuous surveillance for the sake of consumers’ health. The main issue when a food‐borne pathogen outbreak occurs is represented by the identification of the source(s) of contamination. Delivering this information in a timely manner helps to control the problem, with positive outcomes for everyone, especially for the consumers, whose health is in this way preserved, and for the stakeholders involved in food production and distribution, who could face enormous economic losses if recalls or legal issues occur. Whole genome sequencing (WGS) is a tool recently implemented for the characterisation of isolates and the study of outbreaks because of its higher efficiency and faster results, when compared to traditional typing methods. Lower sequencing costs and the development of many bioinformatic tools helped its spread, and much more attention has been given to its use for outbreak investigation. It is important to reach a certain level of standardisation, though, for ensuring result reproducibility and interoperability. Moreover, nowadays it is possible, if not mandatory for Open Science Practices, to share WGS data in publicly available databases, where raw reads, assembled genomes and their corresponding metadata can be easily found and downloaded. The scope of this Fellowship was to provide the Fellow all the training necessary for successfully integrating genomics to surveillance and risk assessment of food‐borne pathogens from farm to fork.

Research – Frozen Vegetable Processing Plants Can Harbour Diverse Listeria monocytogenes Populations: Identification of Critical Operations by WGS


Frozen vegetables have emerged as a concern due to their association with foodborne outbreaks such as the multi-country outbreak of Listeria monocytogenes serogroup IVb linked to frozen corn. The capacity of L. monocytogenes to colonize food-processing environments is well-known, making the bacteria a real problem for consumers. However, the significance of the processing environment in the contamination of frozen foods is not well established. This study aimed to identify potential contamination niches of L. monocytogenes in a frozen processing plant and characterize the recovered isolates. A frozen vegetable processing plant was monitored before cleaning activities. A total of 78 points were sampled, including frozen vegetables. Environmental samples belonged to food-contact surfaces (FCS); and non-food-contact surfaces (n-FCS). Positive L. monocytogenes samples were found in FCS (n = 4), n-FCS (n = 9), and the final product (n = 1). A whole-genome sequencing (WGS) analysis revealed two clusters belonging to serotypes 1/2a-3a and 1/2b-3b). The genetic characterization revealed the presence of four different sequence types previously detected in the food industry. The isolate obtained from the final product was the same as one isolate found in n-FCS. A multi-virulence-locus sequence typing (MVLST) analysis showed four different virulence types (VT). The results obtained highlight the relevant role that n-FCS such as floors and drains can play in spreading L. monocytogenes contamination to the final product. View Full-Text

Research – Invasive listeriosis outbreaks and salmon products: a genomic, epidemiological study


Invasive listeriosis, caused by Listeria (L.) monocytogenes, is a severe foodborne infection, especially for immunocompromised individuals. The aim of our investigation was the identification and analysis of listeriosis outbreaks in Germany with smoked and graved salmon products as the most likely source of infection using whole-genome sequencing (WGS) and patient interviews.

In a national surveillance program, WGS was used for subtyping and core genome multi locus sequence typing (cgMLST) for cluster detection of L. monocytogenes isolates from listeriosis cases as well as food and environmental samples in Germany. Patient interviews were conducted to complement the molecular typing.

We identified 22 independent listeriosis outbreaks occurring between 2010 and 2021 that were most likely associated with the consumption of smoked and graved salmon products. In Germany, 228 cases were identified, of 50 deaths reported (22%) 17 were confirmed to have died from listeriosis. Many of these 22 outbreaks were cross-border outbreaks with further cases in other countries.

This report shows that smoked and graved salmon products contaminated with L. monocytogenes pose a serious risk for listeriosis infection in Germany. Interdisciplinary efforts including WGS and epidemiological investigations were essential to identifying the source of infection. Uncooked salmon products are high risk foods frequently contaminated with L. monocytogenes. In order to minimise the risk of infection for consumers, food producers need to improve hygiene measures and reduce the entry of pathogens into food processing. Furthermore, susceptible individuals should be better informed of the risk of acquiring listeriosis from consuming smoked and graved salmon products’.

Research – Multistate Outbreak Investigation of Salmonella Infections Linked to Kratom: A Focus on Traceback, Laboratory, and Regulatory Activities

Journal of Food Protection

During the spring of 2018, the U.S. Food and Drug Administration (FDA), U.S. Centers for Disease Control and Prevention (CDC), states and local public health agencies responded to a multistate outbreak of gastrointestinal illnesses caused by multiple   Salmonella   serovars and associated with consumption of kratom, a product harvested from a tropical tree native to Southeast Asia. The outbreak included 199 case-patients reported by 41 U.S. states, with illness onset dates ranging from January 11, 2017 to May 8, 2018, leading to 54 hospitalizations, and no deaths. Case-patients reported purchasing kratom products from physical and online retail points of service (POS). Products distributed to 16 POS where 24 case-patients from 17 states purchased kratom were selected for traceback investigation. Traceback revealed that the kratom was imported from several countries, the most common being Indonesia. Local and state officials collected product samples from case-patients and retail POS. The FDA collected 76 product samples from POS and distributors, of which 42 (55%) tested positive for   Salmonella  . The positive samples exhibited a wide range of pulsed field gel electrophoresis (PFGE) patterns and whole genome sequence (WGS) genetic heterogeneity, and a total 25 of 42 (60%) yielded at least one isolate indistinguishable from one or more outbreak-related clinical isolates. While it does not exclude a possibility of a single contamination source, the extent of genetic diversity exhibited by the   Salmonella   isolates recovered from product samples and a lack of traceback convergence, suggested that kratom was widely contaminated across multiple sites from which it was grown, harvested, and packaged. As a result of the contamination, kratom products were recalled by numerous firms (both voluntarily and mandatory). Epidemiologic, traceback, and laboratory evidence supported the conclusion that kratom products were associated with illnesses.

CPS Funded Projects 2022 – Microbiology of Irrigation Water


Microbial characterization of irrigation waters using rapid, inexpensive and portable next generation sequencing technologies

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.

Denmark – Listeria outbreak has sickened 9 in Denmark

Food Safety News

Danish officials are investigating a years-long outbreak of Listeria infections that has affected nine people from 2018 through this month.

The Statens Serum Institut (SSI), Danish Veterinary and Food Administration (Fødevarestyrelsen) and DTU Food Institute are trying to find the source of the infections.

The same type of Listeria monocytogenes has been found in nine people from late 2018 to November 2021. The four patients this year have all only recently become ill. Two people fell sick in both 2018 and 2019 and one case was recorded in 2020.

Among those sick are seven women aged from 35 to 95 years old and two children younger than age 5. They live all over the country. All nine Danish patients have been hospitalized but there have not been any deaths.

Whole genome sequencing (WGS) showed samples from outbreak patients to be closely related, which means it is likely they came from the same source.

Research – Application of Whole Genome Sequencing to Understand Diversity and Presence of Genes Associated with Sanitizer Tolerance in Listeria monocytogenes from Produce Handling Sources


Recent listeriosis outbreaks linked to fresh produce suggest the need to better understand and mitigate L. monocytogenes contamination in packing and processing environments. Using whole genome sequencing (WGS) and phenotype screening assays for sanitizer tolerance, we characterized 48 L. monocytogenes isolates previously recovered from environmental samples in five produce handling facilities. Within the studied population there were 10 sequence types (STs) and 16 cgMLST types (CTs). Pairwise single nucleotide polymorphisms (SNPs) ranged from 0 to 3047 SNPs within a CT, revealing closely and distantly related isolates indicative of both sporadic and continuous contamination events within the facility. Within Facility 1, we identified a closely related cluster (0–2 SNPs) of isolates belonging to clonal complex 37 (CC37; CT9492), with isolates recovered during sampling events 1-year apart and in various locations inside and outside the facility. The accessory genome of these CC37 isolates varied from 94 to 210 genes. Notable genetic elements and mutations amongst the isolates included the bcrABC cassette (2/48), associated with QAC tolerance; mutations in the actA gene on the Listeria pathogenicity island (LIPI) 1 (20/48); presence of LIPI-3 (21/48) and LIPI-4 (23/48). This work highlights the potential use of WGS in tracing the pathogen within a facility and understanding properties of L. monocytogenes in produce settings. View Full-Text

Research -Application of Whole Genome Sequencing to Aid in Deciphering the Persistence Potential of Listeria monocytogenes in Food Production Environments


Listeria monocytogenes is the etiological agent of listeriosis, a foodborne illness associated with high hospitalizations and mortality rates. This bacterium can persist in food associated environments for years with isolates being increasingly linked to outbreaks. This review presents a discussion of genomes of Listeria monocytogenes which are commonly regarded as persisters within food production environments, as well as genes which are involved in mechanisms aiding this phenotype. Although criteria for the detection of persistence remain undefined, the advent of whole genome sequencing (WGS) and the development of bioinformatic tools have revolutionized the ability to find closely related strains. These advancements will facilitate the identification of mechanisms responsible for persistence among indistinguishable genomes. In turn, this will lead to improved assessments of the importance of biofilm formation, adaptation to stressful conditions and tolerance to sterilizers in relation to the persistence of this bacterium, all of which have been previously associated with this phenotype. Despite much research being published around the topic of persistence, more insights are required to further elucidate the nature of true persistence and its implications for public health.