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Next-Generation Sequencing and Emerging Technologies for the Identification and Control...
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Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain

A special issue of Foods (ISSN 2304-8158). This special issue belongs to the section "Food Engineering and Technology".

Deadline for manuscript submissions: closed (15 March 2021) | Viewed by 28365

Special Issue Editor

Dr. Sílvia Bofill-Mas

E-Mail Website
Guest Editor
Laboratory of Viruses Contaminants of Water and Food, Genetics, Microbiology and Statistics Department of the Biology Faculty, University of Barcelona, Barcelona, Spain
Interests: virus; metagenomic studies; next-generation sequencing; microbial contaminants; viral detection techniques; food safety

Special Issue Information

Dear Colleagues,

Food-borne diseases remain a significant cause of illness worldwide. From a microbiological point of view, food can be a vehicle for protozoan, bacterial, and viral infections.

Food contamination can occur at several stages of food chain production, from the irrigation and collection stages on farms to contamination during food processing in industrial settings, food preparation at a restaurant, or at home. Countries use legislation to measure the microbiological quality of water and food, yet food-borne outbreaks are still reported.

In outbreak investigations, fast and reliable tools are required to identify the source of infection. Next-generation sequencing techniques (NGS), including whole-genome sequencing (WGS), that allow the identification of antibiotic resistance genes and the characterization of emergent and new microbial pathogens, have enormous potential as a tool in food safety.

The focus of this Special Issue is to provide more information on the applicability of NGS techniques in the food safety field.

Dr. Sílvia Bofill-Mas
Guest Editor

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Next-generation sequencing (NGS)
  • Whole-genome sequencing (WGS)
  • Food-borne pathogens
  • Food-safety
  • Emergent pathogens
  • Antibiotic resistance

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Published Papers (6 papers)

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Research

21 pages, 1345 KiB  
Article
NGS Techniques Reveal a High Diversity of RNA Viral Pathogens and Papillomaviruses in Fresh Produce and Irrigation Water
by Marta Itarte, Sandra Martínez-Puchol, Eva Forés, Ayalkibet Hundesa, Natàlia Timoneda, Sílvia Bofill-Mas, Rosina Girones and Marta Rusiñol
Foods 2021, 10(8), 1820; https://doi.org/10.3390/foods10081820 - 6 Aug 2021
Cited by 14 | Viewed by 3338
Abstract
Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide [...] Read more.
Fresh fruits and vegetables are susceptible to microbial contamination at every stage of the food production chain, and as a potential source of pathogens, irrigation water quality is a critical factor. Next-generation sequencing (NGS) techniques have been flourishing and expanding to a wide variety of fields. However, their application in food safety remains insufficiently explored, and their sensitivity requires improvement. In this study, quantitative polymerase chain reaction (qPCR) assays showed low but frequent contamination of common circulating viral pathogens, which were found in 46.9% of samples of fresh produce: 6/12 lettuce samples, 4/12 strawberries samples, and 5/8 parsley samples. Furthermore, the application of two different NGS approaches, target enrichment sequencing (TES) for detecting viruses that infect vertebrates and amplicon deep sequencing (ADS), revealed a high diversity of viral pathogens, especially Norovirus (NoV) and Human Papillomavirus (HPV), in fresh produce and irrigation water. All NoV and HPV types found in fresh fruit and vegetable samples were also detected in irrigation water sources, indicating that these viruses are common circulating pathogens in the population and that irrigation water may be the most probable source of viral pathogens in food samples. Full article
(This article belongs to the Special Issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain)
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17 pages, 765 KiB  
Article
Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk
by Ondrej Holý, Julio Parra-Flores, Sarah Lepuschitz, María Paula Alarcón-Lavín, Ariadnna Cruz-Córdova, Juan Xicohtencatl-Cortes, Jetsi Mancilla-Rojano, Werner Ruppitsch and Stephen Forsythe
Foods 2021, 10(1), 20; https://doi.org/10.3390/foods10010020 - 23 Dec 2020
Cited by 19 | Viewed by 4031
Abstract
Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in [...] Read more.
Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in vitro putative virulence factors of six Cronobacter sakazakii strains isolated from powdered milk (PM) in the Czech Republic. Strains were identified by MALDI-TOF MS and whole-genome sequencing (WGS). Virulence and resistance genes were detected with the Ridom SeqSphere+ software task template and the Comprehensive Antibiotic Resistance Database (CARD) platform. Adherence and invasion ability were performed using the mouse neuroblastoma (N1E-115 ATCCCRL-2263) cell line. The CRISPR-Cas system was searched with CRISPRCasFinder. Core genome MLST identified four different sequence types (ST1, ST145, ST245, and ST297) in six isolates. Strains 13755-1B and 1847 were able to adhere in 2.2 and 3.2 × 106 CFU/mL, while 0.00073% invasion frequency was detected only in strain 1847. Both strains 13755-1B and 1847 were positive for three (50.0%) and four virulence genes, respectively. The cpa gene was not detected. Twenty-eight genes were detected by WGS and grouped as flagellar or outer membrane proteins, chemotaxis, hemolysins, and invasion, plasminogen activator, colonization, transcriptional regulator, and survival in macrophages. The colistin-resistance-encoding mcr-9.1 and cephalothin-resis-encoding blaCSA genes and IncFII(pECLA) and IncFIB(pCTU3) plasmids were detected. All strains exhibited CRISPR matrices and four of them two type I-E and I-F matrices. Combined molecular methodologies improve Cronobacter spp. decision-making for health authorities to protect the population. Full article
(This article belongs to the Special Issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain)
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18 pages, 3311 KiB  
Article
The Unexplored Virome of Two Atlantic Coast Fish: Contribution of Next-Generation Sequencing to Fish Virology
by Andreia Filipa-Silva, Ricardo Parreira, Sandra Martínez-Puchol, Sílvia Bofill-Mas, Maria Teresa Barreto Crespo and Mónica Nunes
Foods 2020, 9(11), 1634; https://doi.org/10.3390/foods9111634 - 9 Nov 2020
Cited by 21 | Viewed by 4599
Abstract
Much of the knowledge on viruses is focused on those that can be propagated using cell-cultures or that can cause disease in humans or in economically important animals and plants. However, this only reflects a small portion of the virosphere. Therefore, in this [...] Read more.
Much of the knowledge on viruses is focused on those that can be propagated using cell-cultures or that can cause disease in humans or in economically important animals and plants. However, this only reflects a small portion of the virosphere. Therefore, in this study, we explore by targeted next-generation sequencing, how the virome varies between Atlantic horse mackerels and gilthead seabreams from fisheries and aquaculture from the center and south regions of Portugal. Viral genomes potentially pathogenic to fish and crustaceans, as well as to humans, were identified, namely Astroviridae, Nodaviridae, Hepadnaviridae, Birnaviridae, Caliciviridae, and Picornaviridae families. Also bacteriophages sequences were identified corresponding to the majority of sequences detected, with Myoviridae, Podoviridae, and Siphoviridae, the most widespread families in both fish species. However, these findings can also be due to the presence of bacteria in fish tissues, or even to contamination. Overall, seabreams harbored viruses from a smaller number of families in comparison with mackerels. Therefore, the obtained data show that fish sold for consumption can harbor a high diversity of viruses, many of which are unknown, reflecting the overall uncharacterized virome of fish. While cross-species transmission of bonafide fish viruses to humans is unlikely, the finding of human pathogenic viruses in fish suggest that fish virome can be a potential threat regarding food safety. Full article
(This article belongs to the Special Issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain)
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21 pages, 2579 KiB  
Article
The Benefits of Whole Genome Sequencing for Foodborne Outbreak Investigation from the Perspective of a National Reference Laboratory in a Smaller Country
by Stéphanie Nouws, Bert Bogaerts, Bavo Verhaegen, Sarah Denayer, Florence Crombé, Klara De Rauw, Denis Piérard, Kathleen Marchal, Kevin Vanneste, Nancy H. C. Roosens and Sigrid C. J. De Keersmaecker
Foods 2020, 9(8), 1030; https://doi.org/10.3390/foods9081030 - 1 Aug 2020
Cited by 22 | Viewed by 5129
Abstract
Gradually, conventional methods for foodborne pathogen typing are replaced by whole genome sequencing (WGS). Despite studies describing the overall benefits, National Reference Laboratories of smaller countries often show slower uptake of WGS, mainly because of significant investments required to generate and analyze data [...] Read more.
Gradually, conventional methods for foodborne pathogen typing are replaced by whole genome sequencing (WGS). Despite studies describing the overall benefits, National Reference Laboratories of smaller countries often show slower uptake of WGS, mainly because of significant investments required to generate and analyze data of a limited amount of samples. To facilitate this process and incite policy makers to support its implementation, a Shiga toxin-producing Escherichia coli (STEC) O157:H7 (stx1+, stx2+, eae+) outbreak (2012) and a STEC O157:H7 (stx2+, eae+) outbreak (2013) were retrospectively analyzed using WGS and compared with their conventional investigations. The corresponding results were obtained, with WGS delivering even more information, e.g., on virulence and antimicrobial resistance genotypes. Besides a universal, all-in-one workflow with less hands-on-time (five versus seven actual working days for WGS versus conventional), WGS-based cgMLST-typing demonstrated increased resolution. This enabled an accurate cluster definition, which remained unsolved for the 2013 outbreak, partly due to scarce epidemiological linking with the suspect source. Moreover, it allowed detecting two and one earlier circulating STEC O157:H7 (stx1+, stx2+, eae+) and STEC O157:H7 (stx2+, eae+) strains as closely related to the 2012 and 2013 outbreaks, respectively, which might have further directed epidemiological investigation initially. Although some bottlenecks concerning centralized data-sharing, sampling strategies, and perceived costs should be considered, we delivered a proof-of-concept that even in smaller countries, WGS offers benefits for outbreak investigation, if a sufficient budget is available to ensure its implementation in surveillance. Indeed, applying a database with background isolates is critical in interpreting isolate relationships to outbreaks, and leveraging the true benefit of WGS in outbreak investigation and/or prevention. Full article
(This article belongs to the Special Issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain)
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13 pages, 1751 KiB  
Article
Comparison of Antimicrobial Resistance and Pan-Genome of Clinical and Non-Clinical Enterococcus cecorum from Poultry Using Whole-Genome Sequencing
by Poonam Sharma, Sushim K. Gupta, John B. Barrett, Lari M. Hiott, Tiffanie A. Woodley, Subhashinie Kariyawasam, Jonathan G. Frye and Charlene R. Jackson
Foods 2020, 9(6), 686; https://doi.org/10.3390/foods9060686 - 26 May 2020
Cited by 12 | Viewed by 4096
Abstract
Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial [...] Read more.
Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial resistance genes as well as the pan-genome among the isolates. Eighteen strains selected from our previous study were subjected to WGS using Illumina MiSeq and comparatively analyzed. Assembled contigs were analyzed for resistance genes using ARG-ANNOT. Resistance to erythromycin was mediated by ermB, ermG, and mefA, in clinical isolates and ermB and mefA, in non-clinical isolates. Lincomycin resistance genes were identified as linB, lnuB, lnuC, and lnuD with lnuD found only in non-clinical E. cecorum; however, lnuB and linB were found in only one clinical isolate. For both groups of isolates, kanamycin resistance was mediated by aph3-III, while tetracycline resistance was conferred by tetM, tetO, and tetL. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol, suggesting possible new mechanisms of resistance to these drugs. A comparison of WGS results confirmed that non-clinical isolates contained more resistance genes than clinical isolates. The pan-genome of clinical and non-clinical isolates resulted in 3651 and 4950 gene families, respectively, whereas the core gene sets were comprised of 1559 and 1534 gene families in clinical and non-clinical isolates, respectively. Unique genes were found more frequently in non-clinical isolates than clinical. Phylogenetic analysis of the isolates and all the available complete and draft genomes showed no correlation between healthy and diseased poultry. Additional genomic comparison is required to elucidate genetic factors in E. cecorum that contribute to disease in poultry. Full article
(This article belongs to the Special Issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain)
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14 pages, 2609 KiB  
Article
High-Throughput 16S rRNA Sequencing to Assess Potentially Active Bacteria and Foodborne Pathogens: A Case Example in Ready-to-Eat Food
by Marina Mira Miralles, Lucia Maestre-Carballa, Monica Lluesma-Gomez and Manuel Martinez-Garcia
Foods 2019, 8(10), 480; https://doi.org/10.3390/foods8100480 - 11 Oct 2019
Cited by 18 | Viewed by 6082
Abstract
Technologies to detect the entire bacterial diversity spectra and foodborne pathogens in food represent a fundamental advantage in the control of foodborne illness. Here, we applied high-throughput 16S rRNA sequencing of amplicons obtained by PCR and RT-PCR from extracted DNA and RNA targeting [...] Read more.
Technologies to detect the entire bacterial diversity spectra and foodborne pathogens in food represent a fundamental advantage in the control of foodborne illness. Here, we applied high-throughput 16S rRNA sequencing of amplicons obtained by PCR and RT-PCR from extracted DNA and RNA targeting the entire bacterial community and the active bacterial fraction present in some of the most consumed and distributed ready-to-eat (RTE) salad brands in Europe. Customer demands for RTE food are increasing worldwide along with the number of associated foodborne illness and outbreaks. The total aerobic bacterial count in the analyzed samples was in the range of 2–4 × 106 CFU/g (SD ± 1.54 × 106). Culture validated methods did not detect Salmonella spp., Escherichia coli, and other fecal coliforms. 16S rRNA gene Illumina next-generation sequencing (NGS) data were congruent with these culture-based results and confirmed that these and other well-known foodborne bacterial pathogens, such as Listeria, were not detected. However, the fine-resolution of the NGS method unveiled the presence of the opportunistic pathogens Aeromonas hydrophyla and Rahnella aquatilis (relative frequency of 1.33–7.33%) that were metabolically active in addition to non-pathogenic, active members of Yersinia spp. (relative frequency of 0.0015–0.003%). The common ail and foxA marker genes of Yersinia enterocolitica were not detected by qPCR. Finally, our NGS data identified to non-pathogenic Pseudomonas spp. as the most abundant and metabolically active bacteria in the analyzed RTE salads (53–75% of bacterial abundance). Our data demonstrate the power of sequencing, in parallel, both 16S rRNA and rDNA to identify and discriminate those potentially and metabolically active bacteria and pathogens to provide a more complete view that facilitates the control of foodborne diseases, although further work should be conducted to determine the sensitivity of this method for targeting bacteria Full article
(This article belongs to the Special Issue Next-Generation Sequencing and Emerging Technologies for the Identification and Control of Microbial Contaminants in the Food Chain)
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