Category Archives: WGS

Research – Whole-Genome Sequence Comparisons of Listeria monocytogenes Isolated from Meat and Fish Reveal High Inter- and Intra-Sample Diversity

MDPI

Interpretation of whole-genome sequencing (WGS) data for foodborne outbreak investigations is complex, as the genetic diversity within processing plants and transmission events need to be considered. In this study, we analyzed 92 food-associated Listeria monocytogenes isolates by WGS-based methods. We aimed to examine the genetic diversity within meat and fish production chains and to assess the applicability of suggested thresholds for clustering of potentially related isolates. Therefore, meat-associated isolates originating from the same samples or processing plants as well as fish-associated isolates were analyzed as distinct sets. In silico serogrouping, multilocus sequence typing (MLST), core genome MLST (cgMLST), and pangenome analysis were combined with screenings for prophages and genetic traits. Isolates of the same subtypes (cgMLST types (CTs) or MLST sequence types (STs)) were additionally compared by SNP calling. This revealed the occurrence of more than one CT within all three investigated plants and within two samples. Analysis of the fish set resulted in predominant assignment of isolates from pangasius catfish and salmon to ST2 and ST121, respectively, potentially indicating persistence within the respective production chains. The approach not only allowed the detection of distinct subtypes but also the determination of differences between closely related isolates, which need to be considered when interpreting WGS data for surveillance.

USA – Outbreak Investigation of Salmonella: Seafood (October 2022)

FDA

The FDA, along with CDC and state and local partners, is investigating a multistate outbreak of Salmonella Litchfield infections linked to fresh, raw salmon supplied to restaurants in California and Arizona by Mariscos Bahia, Inc.

Based on epidemiologic information provided by CDC and interviews conducted by state and local public health officials, of 16 people interviewed, 12 reported eating sushi, sashimi, or poke. Of those interviewed, 11 people remembered details about the type of fish consumed and 9 report eating raw salmon before getting sick. The FDA’s investigation traced the distribution of fresh, raw salmon back to Mariscos Bahia, Inc.

In addition, the FDA collected an environmental sample that included multiple swabs at Mariscos Bahia, Inc. (Pico Rivera, CA). Multiple environmental swabs collected at the facility are positive for Salmonella and subsequent  Whole Genome Sequencing (WGS) analysis is ongoing. The WGS completed to date indicates the Salmonella detected in at least one of the swabs from the facility matches the outbreak strain. While epidemiological evidence indicates that ill people consumed fresh, raw salmon processed at this firm, the presence of Salmonella in the processing environment indicates that additional types of fish processed in the same area of the facility could also be contaminated which includes fresh, raw halibut, Chilean seabass, tuna, and swordfish. Salmon, halibut, Chilean seabass, tuna, and swordfish processed in Marisco Bahia Inc.’s Pico Rivera, CA, facility could have also been sent to the Mariscos Bahia, Inc. facilities in Phoenix, AZ and then sent to restaurants.

The firm is cooperating with the FDA investigation and has agreed to initiate a voluntary recall. As a part of the firm’s voluntary recall, the firm will contact its direct customers who received recalled product.

The FDA’s investigation is ongoing. Updates to this advisory will be provided as they become available.

Recommendation

According to Mariscos Bahia, Inc., seafood was only sold directly to restaurants in California and Arizona and would not be available for purchase by consumers in stores.

Restaurants should check with their suppliers and not sell or serve salmon, halibut, Chilean seabass, tuna, and swordfish received fresh, not frozen from Mariscos Bahia, Inc. (Pico Rivera, CA and Phoenix, AZ) on or after June 14, 2022. If restaurants received these fish and then froze it, they should not sell or serve it. Restaurants should also be sure to wash and sanitize locations where these fish from Mariscos Bahia, Inc. were stored or prepared.

Consumers eating salmon, halibut, Chilean seabass, tuna, and swordfish at a restaurant in California or Arizona should ask whether the fish is from Mariscos Bahia, Inc and was received fresh, not frozen.


Map of U.S. Distribution

Map of U.S. Distribution

Case Count Map Provided by CDC

Salmon Outbreak of Salmonella - CDC Case Count Map as of October 19, 2022

Case Counts

Total Illnesses: 33
Hospitalizations: 13
Deaths: 0
Last illness onset: September 18, 2022
States with Cases: AZ (11), CA (21), IL (1)
Product Distribution*:  AZ, CA
*Distribution has been confirmed for states listed, but product could have been distributed further, reaching additional states

Research – Phenotypic and genotypic characterization of salmonella Enteritidis isolated from two consecutive Food-Poisoning outbreaks in Sichuan, China

Wiley Online

Abstract

Salmonella enterica serotype Enteritidis (SE) is a primary pathogen that causes foodborne diseases in humans. Although whole-genome sequencing (WGS) -based typing analyses have been increasingly used to investigate food-poisoning outbreaks, they are rarely applied to the epidemiology of multiple Salmonella outbreaks in Sichuan, China. This study therefore isolated SE from patients and food of two consecutive food-poisoning outbreaks during 2020 in Sichuan, China. We tracked outbreak origin using epidemiological investigation, serotyping, antimicrobial susceptibility testing (AST), pulsed-field gel electrophoresis (PFGE), and WGS. We also determined phylogenetic relationships using PFGE, whole and core genome multilocus sequence typing (wg/cgMLST), and whole-genome single nucleotide polymorphism (wgSNP) analyses. Epidemiological investigations identified a correlation between cake consumption and food poisoning. Thirteen strains isolated from patients and one strain isolated from the cake were confirmed as SE. Among the 14 strains, only six shared the same AST pattern (AMP-AMS-Sul-STR). Isolates from patients and cakes were indistinguishable in PFGE results. All four methods, namely PFGE, wgMLST, cgMLST, and wgSNP were appropriate for bacterial typing in SE-related outbreak investigation. However, wgSNP can assign 12 SE strains from the first outbreak to one cluster and assign two SE strains from the second outbreak to another cluster, while PFGE, wgMLST, cgMLST did not successfully distinguish the SE strains from different outbreaks. Thus, we conclude that SNP-based phylogenetic analysis might be a viable method for differentiating SE strains at the outbreak level.

Research – Whole-Genome Analysis of Staphylococcus aureus Isolates from Ready-to-Eat Food in Russia

MDPI

This study provides a thorough investigation of a diverse set of antimicrobial resistant (AMR) Staphylococcus aureus isolates collected from a broad range of ready-to-eat (RTE) food in various geographic regions of Russia ranging from Pskov to Kamchatka. Thirty-five isolates were characterized using the whole genome sequencing (WGS) analysis in terms of clonal structure, the presence of resistance and virulence determinants, as well as plasmid replicon sequences and CRISPR/Cas systems. To the best of our knowledge, this is the first WGS-based surveillance of Russian RTE food-associated S. aureus isolates. The isolates belonged to fifteen different multilocus sequence typing (MLST)-based types with a predominant being the ones of clonal complex (CC) 22. The isolates studied can pose a threat to public health since about 40% of the isolates carried at least one enterotoxin gene, and 70% of methicillin-resistant (MRSA) isolates carried a tsst1 gene encoding a toxin that may cause severe acute disease. In addition, plasmid analysis revealed some important characteristics, e.g., Rep5 and Rep20 plasmid replicons were a “signature” of MRSA CC22. By analyzing the isolates belonging to the same/single strain based on cgMLST analysis, we were able to identify the differences in their accessory genomes marking their dynamics and plasticity. This data is very important since S. aureus isolates studied and RTE food, in general, represent an important route of transmission and dissemination of multiple pathogenic determinants. We believe that the results obtained will facilitate performing epidemiological surveillance and developing protection measures against this important pathogen in community settings. View Full-Text

USA – FDA Issues Country-Wide Import Alert for Enoki Mushrooms from the Republic of Korea – Listeria monocytogenes

FDA

The FDA announced today that its Import Divisions may detain without physical examination, importations of enoki mushrooms from the ROK. This country-wide import alert, IA #25-21, “Detention Without Physical Examination of Enoki Mushrooms from Korea (the Republic of) due to Listeria monocytogenes,” was issued to protect public health and help prevent the importation of enoki mushrooms that could be linked to human infections. The FDA issues import alerts to help prevent potentially violative products from being distributed in the United States.

In fiscal year 2021, FDA testing revealed that 43% of enoki mushrooms sampled from the ROK were contaminated with Listeria monocytogenes (L. monocytogenes). L. monocytogenes is a human pathogen that can be found in moist environments, soil, water, decaying vegetation and animals, and can survive and even grow under refrigeration and other food preservation measures.

The sampling was conducted following an FDA investigation into a multistate outbreak spanning from 2016-2020. This outbreak linked multiple cases of human infections of L. monocytogenes to enoki mushrooms from the ROK. The outbreak accounted for a total of 36 U.S. cases reported across 17 states, 12 cases in Canada, and six cases in Australia. The 36 U.S. cases yielded 31 hospitalizations and four deaths.

From March 2020 through May 2022, state public health authorities conducted sampling of enoki mushrooms from U.S. retail locations. L. monocytogenes was detected in multiple state samples, which led to 21 recalls of enoki mushrooms in the United States. Nine of the recalls were linked to enoki mushrooms grown in the ROK and were confirmed by labelling, traceback, or whole genome sequencing (WGS).

After the 2020 outbreak, the FDA began implementing an Imported Specialty Mushroom Prevention Strategy, with a focus on enoki mushrooms, to protect public health and prevent future L. monocytogenes outbreaks in specialty imported mushrooms. The FDA’s prevention strategies are affirmative, deliberate approaches undertaken by the agency to limit or prevent the recurrence of a root cause that led to an outbreak or adverse incident.

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

MDPI

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

EFSA

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

MDPI

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

Tandfonline

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’.